CTSP cancer-testis antigens

ABSTRACT

The invention relates to CTSP polypeptides and the nucleic acid molecules that encode them. The invention further relates to the use of the nucleic acid molecules, polypeptides and fragments thereof in methods and compositions for the diagnosis, prognosis and treatment of diseases, such as cancer. More specifically, the invention relates to the discovery of a novel cancer/testis (CT) antigen, CTSP-1.

Related Application

This application is a divisional of U.S. application Ser. No.12/087,954, filed on Oct. 24, 2008, now U.S. Pat. No. 8,207,300, whichis a national stage filing under 35 U.S.C. 371 of InternationalApplication PCT/US2007/002486, filed Jan. 29, 2007, which claims thebenefit under 35 U.S.C. §119(e) of United States provisional patentapplication Ser. No. 60/763345, filed Jan. 30, 2006, the contents ofwhich are herein incorporated by reference in their entirety

FIELD OF THE INVENTION

The invention relates to a family of cancer-testis antigens and thenucleic acid molecules that encode them. The invention further relatesto the use of the nucleic acid molecules, polypeptides and fragmentsthereof in methods and compositions for the diagnosis, prognosis andtreatment of diseases, such as cancer. More specifically, the inventionrelates to the discovery of a novel cancer/testis (CT) antigen, CTSP-1.

BACKGROUND OF THE INVENTION

CT antigens are predominantly expressed in normal gametogenic tissues aswell as in different histological types of tumors (Scanlan et al., 2002,Immunol Rev. 188:22-32. Review; Scanlan et al., 2004, Cancer Immun. 4:1.Review; Simpson et al., 2005, Nat Rev Cancer. 5(8):615-25. Review;Zendman et al., 2003, J Cell Physiol. 194(3):272-88. Review). In testis,CT antigens are expressed exclusively in cells of the germ cell lineage,although there is a marked variation in the protein expression patternduring different stages of sperm development. Likewise, a heterogeneousexpression is also observed in tumors (Scanlan et al., 2002, ImmunolRev. 188:22-32. Review; Zendman et al., 2003, J Cell Physiol.194(3):272-88. Review). Methylation status of the promoter region seemsto be the main, but not the only regulator of their specific expressionpattern (Scanlan et al., 2002, Immunol Rev. 188:22-32. Review; Simpsonet al., 2005, Nat Rev Cancer. 5(8):615-25. Review; Zendman et al., 2003,J Cell Physiol. 194(3):272-88. Review). Most CT antigens have no definedbiological function but their involvement in signaling, transcription,translation and chromosomal recombination has been proposed (Simpson etal., 2005, Nat Rev Cancer. 5(8):615-25. Review; Zendman et al., 2003, JCell Physiol. 194(3):272-88. Review). It has also been proposed that theaberrant expression of CT antigens in tumors recapitulates portions ofthe germline gene expression programme and is related to somecharacteristics of the neoplastic phenotype such as immortality,invasiveness, immune evasion and metastatic capacity (Simpson et al.,2005, Nat Rev Cancer. 5(8):615-25. Review; Old, L J., 2001, CancerImmun. 1: 1).

Due to their restricted expression pattern, CT antigens are consideredto be ideal targets for cancer immunotherapy (Scanlan et al., 2002,Immunol Rev. 188:22-32. Review; Bodey, 2002, Expert Opin Biol Ther.2(6):577-84. Review). Indeed, a small subset of patients immunized withthe known CT antigens MAGE-A and NY-ESO-1 have shown clinical benefitsfollowing immunization (Chen et al., 2004, Proc. Natl. Acad. Sci. U.S.A.101: 9363-9368; Davis et al., 2004, Proc. Natl. Acad. Sci. U.S.A.101:10697-10702; Jager et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:12198-12203; Marchand et al., 1999, Int. J. Cancer. 80: 219-230).However, because CT antigens are expressed in only a small subset ofhuman tumors and in only a fraction of cases of a given tumor type, theidentification of additional CT antigens is crucial for improvingcurrent immunotherapy protocols. Presently, 44 distinct CT-antigenfamilies have been described, of which several have multiple membersresulting in a total of 89 transcripts (Simpson et al., 2005, Nat RevCancer. 5(8):615-25. Review).

The identification of additional CT antigens and other genes having atumor-associated expression profile is needed for the development ofadditional therapeutics and diagnostics to permit effective treatmentand diagnosis of a broader group of cancer patients.

SUMMARY OF THE INVENTION

During the process of identification of novel genes located on humanchromosome 21 (HC21) (Reymond et al., 2002, Genomics 79: 824-832), wefound a gene (C21ORF99) which is predominantly expressed in normaltestis and has a high similarity to the amino-terminal region of thebreast differentiation antigen NYBR-1 (Jager et al., 2001, Cancer Res.61: 2055-2061). In this work, we refined the characterization of theC21ORF99 gene structure and renamed it to CTSP-1. We found that CTSP-1has a restricted expression pattern in normal tissues characteristic ofCT antigens and is expressed at a high frequency in tumor cell lines andin tumor samples. We also found that CTSP-1 is part of a highlyconserved gene family dispersed throughout the genome and that somemembers of this family have also an expression pattern characteristic ofCT antigens. Antibodies against members of this gene family werefrequently detected in plasma samples from patients with a wide spectrumof tumors.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided that is selected from the group consisting of:

(a) complements of nucleic acid molecules that hybridize under highstringency conditions to a second nucleic acid molecule including anucleotide sequence set forth as any of SEQ ID NOs: 1-11, wherein thecomplements exclude SEQ ID NO: 12,

(b) nucleic acid molecules that differ from the nucleic acid moleculesof (a) in codon sequence due to the degeneracy of the genetic code, and

(c) full-length complements of (a) or (b).

In some embodiments, the isolated nucleic acid molecule includes, orpreferably consists of, a nucleotide sequence set forth as any of SEQ IDNOs: 1-11. In preferred embodiments, the isolated nucleic acid moleculeis a CTSP-1 nucleic acid molecule that includes, or preferably consistsof, a nucleotide sequence set forth as any of SEQ ID NOs: 1-8, aprotein-coding portion thereof, or an alternatively spliced productthereof.

According to a second aspect of the invention, isolated nucleic acidmolecules are provide that include, or preferably consist of, one ormore of SEQ ID NOs:43-57 or full-length complements thereof, wherein thenucleic acid molecule does not consist of SEQ ID NO:12.

According to a third aspect of the invention, an isolated nucleic acidmolecule is provided that includes (a) a nucleotide sequence that is atleast about 90% identical to a nucleotide sequence set forth as any ofSEQ ID NOs: 1-11 or a full-length complement thereof, or (b) anucleotide sequence that is at least about 90% identical to a nucleotidesequence set forth as any of SEQ ID NOs:43-57 or a full-lengthcomplement thereof, wherein the nucleotide sequence does not consist ofSEQ ID NO:12.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that is at least about 95% identical, preferably at least about97% identical, more preferably at least about 98% identical, and mostpreferably at least about 99% identical to the nucleotide sequence.

According to a fourth aspect of the invention, compositions are providedthat include any of the foregoing isolated nucleic acid molecules and acarrier, or any of the foregoing isolated nucleic acid moleculesattached to a solid substrate.

According to a fifth aspect of the invention, kits are provided thatinclude one or more nucleic acid molecules that hybridize under highstringency conditions to a nucleotide sequence set forth as any of SEQID NOs:1-11 and 43-57.

In some embodiments, the one or more nucleic acid molecules aredetectably labeled. In other embodiments, the one or more nucleic acidmolecules consist of a first primer and a second primer, wherein thefirst primer and the second primer are constructed and arranged toselectively amplify at least a portion of a nucleic acid molecule thatcomprises a nucleotide sequence set forth as any of SEQ ID NOs:1-11 and43-57. In further embodiments, the one or more nucleic acids are boundto a solid substrate.

According to a sixth aspect of the invention, expression vectorsincluding any of the foregoing isolated nucleic acid molecules, operablylinked to a promoter, are provided. Also provided are isolated hostcells transformed or transfected with the expression vectors. In someembodiments, the host cells express a MHC molecule, preferablyrecombinantly. In some preferred embodiments, the host cell is adendritic cell.

Also provided in accordance with the invention are compositions thatinclude the foregoing isolated host cells and a carrier.

According to a seventh aspect of the invention, isolated polypeptidesencoded by any of the foregoing isolated nucleic acid molecules areprovided, as are fragments of the polypeptides that are at least eightamino acids in length. In some embodiments, the isolated polypeptideincludes, or preferably consists of, an amino acid sequence set forth asany of SEQ ID NOs: 13-23.

According to an eighth aspect of the invention, compositions includingany of the foregoing isolated polypeptides and a carrier are provided;preferably the compositions also include an adjuvant. Also provided arecompositions including any of the foregoing isolated polypeptidesattached to a solid substrate.

In some embodiments, the compositions further include at least oneadditional cancer-testis antigen polypeptide, preferably a NY-ESO-1,MAGE or SSX polypeptide, preferably also containing a carrier and/or anadjuvant.

According to a ninth aspect of the invention, isolated antibodies orantigen-binding fragments thereof are provided that selectively bind tothe foregoing isolated polypeptides. In some embodiments, the antibodyis a monoclonal antibody, a human antibody, a domain antibody, ahumanized antibody, a single chain antibody or a chimeric antibody. Inother embodiments, the isolated antigen-binding fragment thereof ofclaim A 16, wherein the antibody fragment is a F(ab′)₂, Fab, Fd, or Fvfragment. Also provided are compositions including any of the foregoingisolated antibodies or antigen-binding fragments and a carrier, orattached to a solid substrate. Kits including the isolated antibodies orantigen-binding fragments also are provided.

According to a tenth aspect of the invention, methods of diagnosingcancer in a subject are provided. The methods include obtaining abiological sample from the subject, and determining the presence in thebiological sample of an antibody that binds specifically to one or morepolypeptides encoded by a nucleotide sequence set forth as any of SEQ IDNO: 1-11. The presence of the antibody is indicative of the subjecthaving cancer.

In some embodiments, the step of determining the presence of theantibody includes contacting the biological sample with one or morepolypeptides encoded by a nucleic acid molecule comprising (1) anucleotide sequence set forth as any of SEQ ID NOs: 1-11 or (2) anucleotide sequence that is at least 90% identical to the nucleotidesequence of (1), and determining the binding of the antibody to thepolypeptide. In certain embodiments, the nucleic acid molecule includesa nucleotide sequence set forth as any of SEQ ID NO: 1-8. In otherembodiments, the polypeptide includes an amino acid sequence set forthas any of SEQ ID NOs: 13-23, preferably SEQ ID NOS: 13-20, or a fragmentthereof that is at least eight amino acids in length. Preferably thepolypeptide is produced recombinantly and/or is bound to a substrate.

In other embodiments, the step of determining the binding of theantibody with the polypeptide is performed with an ELISA-based method.

In preferred embodiments of the methods, the biological sample is bloodor serum.

According to an eleventh aspect of the invention, methods for diagnosingcancer in a subject are provided. The methods include obtaining abiological sample from a subject, and determining the expression in thebiological sample of a polypeptide or a nucleic acid molecule thatencodes the polypeptide, wherein the nucleic acid molecule includes (1)a nucleotide sequence set forth as any of SEQ ID NOs:1-11 or (2) anucleotide sequence that is at least 90% identical to the nucleotidesequence of (1). The expression in the biological sample of thepolypeptide or the nucleic acid molecule that encodes it is indicativeof the subject having cancer.

In some embodiments, the nucleic acid molecule includes the nucleotidesequence set forth as any of SEQ ID NOs:1-8. In other embodiments, thepolypeptide includes an amino acid sequence set forth as any of SEQ IDNOs: 13-23, preferably SEQ ID NOS: 13-20, or a fragment thereof that isat least eight amino acids in length.

In other embodiments, the step of determining the expression of thepolypeptide or the nucleic acid molecule that encodes the polypeptideincludes contacting the biological sample with an agent that selectivelybinds to the polypeptide or the nucleic acid molecule that encodes thepolypeptide.

In some embodiments, the agent is a nucleic acid probe or a nucleic acidprimer. Optionally, the expression of the nucleic acid molecule isdetermined by nucleic acid hybridization using the nucleic acid probe ornucleic acid amplification using the nucleic acid primer. Preferably thenucleic acid amplification is real-time RT-PCR or RT-PCR. Preferably thenucleic acid hybridization is performed using a nucleic acid microarraycontaining the nucleic acid probe.

In some embodiments, the agent is a polypeptide, preferably an antibodyor antigen-binding fragment thereof, more preferably a monoclonalantibody or a F(ab′)₂, Fab, Fd, or Fv fragment. In certain embodiments,the antibody or antigen-binding fragment is labeled with a detectablelabel, preferably a fluorescent or radioactive label.

In preferred embodiments of the methods, the sample comprises tissue,cells, and/or blood.

According to a twelfth aspect of the invention, methods for determiningonset, progression, or regression of cancer in a subject are provided.The methods include obtaining from a subject a first biological sampleat a first time, determining the expression in the first sample of apolypeptide or a nucleic acid molecule that encodes the polypeptide,wherein the nucleic acid molecule includes (1) a nucleotide sequence setforth as any of SEQ ID NOs:1-11 or (2) a nucleotide sequence that is atleast 90% identical to the nucleotide sequence of (1), obtaining fromthe subject a second biological sample at a second time subsequent tothe first time, determining the expression in the second sample of thepolypeptide or the nucleic acid molecule that encodes the polypeptide,and comparing the expression in the first sample to the expression inthe second sample as a determination of the onset, progression, orregression of the cancer. An increase in expression in the second samplecompared to the first sample is indicative of onset or progression ofthe cancer, and a decrease in the expression in the second samplecompared to the first sample is indicative of regression of the cancer.

In some embodiments, the nucleic acid molecule includes the nucleotidesequence set forth as any of SEQ ID NOs:1-8. In other embodiments, thepolypeptide includes an amino acid sequence set forth as any of SEQ IDNOs: 13-23, preferably SEQ ID NOS: 13-20, or a fragment thereof that isat least eight amino acids in length.

In other embodiments, the step of determining the expression of thepolypeptide or the nucleic acid molecule that encodes the polypeptideincludes contacting first biological sample and the second biologicalsample with an agent that selectively binds to the polypeptide or thenucleic acid molecule that encodes the polypeptide.

In some embodiments, the agent is a nucleic acid probe or a nucleic acidprimer. Optionally, the expression of the nucleic acid molecule isdetermined by nucleic acid hybridization using the nucleic acid probe ornucleic acid amplification using the nucleic acid primer. Preferably thenucleic acid amplification is real-time RT-PCR or RT-PCR. Preferably thenucleic acid hybridization is performed using a nucleic acid microarraycontaining the nucleic acid probe.

In some embodiments, the agent is a polypeptide, preferably an antibodyor antigen-binding fragment thereof, more preferably a monoclonalantibody or a F(ab′)₂, Fab, Fd, or Fv fragment. In certain embodiments,the antibody or antigen-binding fragment is labeled with a detectablelabel, preferably a fluorescent or radioactive label.

In preferred embodiments of the methods, the sample comprises tissue,cells, and/or blood.

According to a thirteenth aspect of the invention, methods for treatingcancer in a subject are provided. The methods include administering tothe subject an agent that stimulates an immune response to a polypeptideencoded by a nucleic acid molecule comprising a nucleotide sequence thatis at least 90% identical to the nucleotide sequence set forth as any ofSEQ ID NOs:1-11.

In some embodiments, the nucleic acid molecule includes the nucleotidesequence set forth as any of SEQ ID NOs:1-11, preferably SEQ ID NOs:1-8.In other embodiments, the polypeptide includes an amino acid sequenceset forth as any of SEQ ID NOs: 13-23, preferably SEQ ID NOS: 13-20, ora fragment thereof that is at least eight amino acids in length.

In some embodiments, the agent that stimulates the immune response is anucleic acid that encodes the polypeptide operably linked to a promoter;the polypeptide; a cell that expresses the polypeptide, preferably acell that also expresses a MHC molecule; a peptide fragment of thepolypeptide; or a complex of a peptide fragment of the polypeptide and aMHC molecule. Optionally the agent further comprises an adjuvant or acytokine.

In some embodiments, the immune response elicited by an agent or agentsaccording to the invention is a B cell response. In some embodiments,the immune response elicited by an agent or agents according to theinvention is a T cell response, preferably a CD4+ T cell and/or CD8+ Tcell response.

According to a fourteenth aspect of the invention, methods for treatingcancer in a subject are provided. The methods include administering to asubject an effective amount of an antibody or antigen-binding fragmentthereof that specifically binds to a polypeptide that) comprises anamino acid sequence set forth as any of SEQ ID NOs: 13-23, or a peptidefragment thereof, or a complex of the peptide fragment and a MHC or HLAmolecule.

In some embodiments, the antibody is a monoclonal antibody, preferably achimeric, human, or humanized antibody, or a single chain antibody. Inother embodiments, the antigen-binding fragment is a F(ab′)₂, Fab, Fd,or Fv fragment.

In still other embodiments, the antibody or antigen-binding fragmentthereof is bound to a cytotoxic agent, preferably one selected from thegroup consisting of: calicheamicin, esperamicin, methotrexate,doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C,cisplatinum, etopside, bleomycin and 5-fluorouracil. In furtherembodiments, the cytotoxic agent is a radioisotope, preferably one thatemits α radiation, β radiation or γ radiation. In particularembodiments, the radioisotope is selected from the group consisting of²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I,¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra and ²²³Ra.

The invention also involves the use of the genes, gene products,fragments thereof, agents which bind thereto, and other compositions andmolecules described herein in the preparation of medicaments. Aparticular medicament is for treating cancer.

Yet another aspect of the invention provides methods for assessing theprognosis of a subject with prostate cancer. Accordingly, these methodsinclude the steps of obtaining one or more biological samples from asubject (e.g., blood, serum, plasma, tissue biopsy, etc.) anddetermining the presence or absence of anti-CTSP-1 antibodies in thesample(s). The absence of anti-CTSP-1 antibodies in the biologicalsample is indicative of poor prognosis (e.g., shortened biochemicalrecurrence-free interval), whereas the presence of anti-CTSP-1antibodies in the biological sample is indicative of good prognosis(e.g., longer survival as compared to subjects without detectableanti-CTSP-1 antibodies).

In some embodiments, the steps of obtaining a biological sample anddetermining the absence or presence of anti-CTSP-1 antibodies in thesample may be repeated one or more times (e.g., about 1-12 months afterthe first sample is obtained).

In some embodiments, the method of assessing the prognosis for a subjectdiagnosed with prostate cancer may further comprise measuring anon-anti-CTSP-1 antibody prostate cancer marker, such as PSA, PSMA andPAP.

Yet in other embodiments, the methods of assessing the prognosis for asubject diagnosed with prostate cancer may further include measuring thelevel or presence of another prostate cancer marker, such as NY-ESO-1.In some cases, the method includes determining the presence or absenceof NY-ESO-1 mRNA and/or NY-ESO-1 polypeptide in a biological sample. Thepresence of NY-ESO-1 mRNA and/or NY-ESO-1 polypeptide in the biologicalsample is indicative of poor prognosis. In some cases, the methodincludes determining the presence or absence anti-NY-ESO-1 antibodies ina biological sample. The presence of anti-NY-ESO-1 antibodies in thebiological sample is indicative of poor prognosis.

These and other aspects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of the strategies used in thegeneration of CTSP-1 consensus sequence. NYBR-1, NYBR-1.1 and C21ORF99sequences were aligned to the genomic sequence of the human chromosome21 (HC21). Exons are represented as boxes and lines correspond tointrons. RT-PCR and RACE primers are represented with arrows andtriangles correspond to polyadenylation signals. Repetitive elements(LTR and Alu) are represented as black boxes.

FIG. 2 provides a schematic representation of the exon/intron structureof the CTSP-1 family members. Complete and partial copies arerepresented with their corresponding chromosomal location and similarityto CTSP-1 consensus sequence. Repetitive elements (LTR and Alu) arerepresented as black boxes and exons are numbered from 1 to 15.

FIG. 3 depicts CTSP-1 alternative splicing isoforms. FIG. 3A provides aSouthern blot of RT-PCR products amplified with CTSP-1 specific primers.Alternative splicing isoforms (a-h) are indicated with arrows. cDNAsamples used were: normal testis (1), A172 glioblastoma cell line (2),A2058 melanoma cell line (3), H1155 lung tumor cell line (4), breasttumor samples (5,6), prostate tumor samples (7,8), no cDNA negativecontrol (9). FIG. 3B provides a schematic representation of CTSP-1alternative splicing isoforms (a-h). Exons are numbered from 1 to 9 andthe coding exons are represented as gray boxes. Primers used in RT-PCRamplifications are represented as arrows and the size of the putativeopen reading frame for each splicing isoform is provided in amino acids(aa).

FIG. 4 depicts CTSP-1 mRNA expression patterns in normal tissues andtumor cell lines using a Southern blot of RT-PCR products amplified withCTSP-1 specific primers. FIG. 4A: Normal cDNA samples used were:1—testis, 2—lung, 3—prostate, 4—small intestine, 5—breast, 6—brain,7—heart, 8—uterus, 9—bone marrow, 10—placenta, 11—colon, 12—fetal brain,13—liver, 14—fetal liver, 15—thymus, 16—salivary gland, 17—spinal cord,18—kidney, 19—spleen, 20—skeletal muscle, 21—adrenal gland, 22—no cDNAnegative control. FIG. 4B: cDNA samples from tumor cell lines used were:1—Caski, 2—Hela, 3—A172, 4—T98G, 5—HL-60, 6—K562, 7—H358, 8—H1155,9—Du145, 10—PC3, 11—SCABER, 12—IM9, 13—FADu, 14—MCF-7, 15—MDA-436,16—MDA-231, 17—SW-480, 18—SAOS-2, 19—A2058, 20—SKmel-25, 21—HEPG2, 22—nocDNA negative control. GAPDH amplification was used as positive controlfor cDNA synthesis.

FIG. 5 depicts CTSP-1 protein expression in normal testis and inprostate tissue. FIG. 5A: CTSP-1 protein was detected by Western blot inprotein extract from normal testis using a CTSP-1 polyclonal antibody(1) and pre-immune serum used as negative control (2). Molecular weightmarkers are indicated (M). FIG. 5B: Immunohistochemistry staining ofCTSP-1 protein in normal testis (1), prostate tumor (3) and normaladjacent prostate tissue (4) using a anti-CTSP-1 antibody. Normal testis(2) incubated with pre-immune sera as negative control.

FIG. 6 is an image of Western blot analysis of antibodies in plasmasamples from cancer patients that are reactive with CTSP-1 recombinantprotein. A Western blot using CTSP-1 recombinant protein and plasmasamples from prostate cancer patients (lanes 1-7). An anti-HisTagantibody was used as positive control in the Western blot experiments(lane 8). Molecular weight markers are indicated in kilodaltons (KDa).

FIG. 7 depicts induction of CTSP-1 gene expression by treatment with ademethylating agent. The MCF-7 breast tumor cell line was treated with5-Aza 2′ deoxycytidine and CTSP-1 expression was detected by RT-PCR.cDNAs used in the RT-PCR amplification were: MCF-7 mock (lanes marked1), MCF-7 treated (lanes marked 2), normal testis as positive control(lanes marked 3) and no cDNA as negative control (lanes marked 4). GAPDHamplification was used as positive control for cDNA synthesis.

FIG. 8 provides two graphs showing survival curves according to thepresence of an anti-CTSP-1 humoral immune response using two parameters:FIG. 8A depicts biochemical-recurrence-free survival; and FIG. 8Bdepicts disease-free survival.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of CTSP-1 variant a (completemRNA sequence, 3915 bp).

SEQ ID NO:2 is the nucleotide sequence of CTSP-1 variant b (without exon5).

SEQ ID NO:3 is the nucleotide sequence of CTSP-1 variant c (without exon4).

SEQ ID NO:4 is the nucleotide sequence of CTSP-1 variant d (without exon7).

SEQ ID NO:5 is the nucleotide sequence of CTSP-1 variant e (withoutexons 4 and 5).

SEQ ID NO:6 is the nucleotide sequence of CTSP-1 variant f (withoutexons 5 and 7).

SEQ ID NO:7 is the nucleotide sequence of CTSP-1 variant g (withoutexons 4 and 7).

SEQ ID NO:8 is the nucleotide sequence of CTSP-1 variant h (withoutexons 4, 5 and 7).

SEQ ID NO:9 is the nucleotide sequence of CTSP-2 (complete mRNAsequence, 3923 bp).

SEQ ID NO:10 is the nucleotide sequence of CTSP-3 (complete mRNAsequence, 3938 bp).

SEQ ID NO:11 is the nucleotide sequence of CTSP-4 (complete mRNAsequence, 3907 bp).

SEQ ID NO:12 is the nucleotide sequence of C21or199 (complete mRNAsequence, 579 bp).

SEQ ID NO:13 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant a (115 aa).

SEQ ID NO:14 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant b (115 aa).

SEQ ID NO:15 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant c (117 aa).

SEQ ID NO:16 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant d (115 aa).

SEQ ID NO:17 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant e (195 aa).

SEQ ID NO:18 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant f (115 aa).

SEQ ID NO:19 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant g (117 aa).

SEQ ID NO:20 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-1 variant h (202 aa). This is the longest ofthe polypeptides encoded by CTSP-1 variants, and was used for generationof CTSP-1 recombinant protein.

SEQ ID NO:21 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-2 (115 aa).

SEQ ID NO:22 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-3 (115 aa).

SEQ ID NO:23 is the amino acid sequence of polypeptide encoded by theopen reading frame of CTSP-4 (115 aa).

SEQ ID NO:24 is the amino acid sequence of polypeptide encoded by theopen reading frame of C21orf99 (78 aa).

SEQ ID NO:25 is the nucleotide sequence of the CTSP-F1 primer.

SEQ ID NO:26 is the nucleotide sequence of the CTSP-R1 primer.

SEQ ID NO:27 is the nucleotide sequence of the CTSP-F2 primer.

SEQ ID NO:28 is the nucleotide sequence of the CTSP-R2 primer.

SEQ ID NO:29 is the nucleotide sequence of the SP-RACE primer.

SEQ ID NO:30 is the nucleotide sequence of the Adaptor primer.

SEQ ID NO:31 is the nucleotide sequence of the SP-RACE-3N primer.

SEQ ID NO:32 is the nucleotide sequence of the Adaptor-N primer.

SEQ ID NO:33 is the nucleotide sequence of the CT1F primer.

SEQ ID NO:34 is the nucleotide sequence of the CT1R primer.

SEQ ID NO:35 is the nucleotide sequence of the CT2F primer.

SEQ ID NO:36 is the nucleotide sequence of the CT2R primer.

SEQ ID NO:37 is the nucleotide sequence of the CT3F primer.

SEQ ID NO:38 is the nucleotide sequence of the CT3R primer.

SEQ ID NO:39 is the nucleotide sequence of the CT4F primer.

SEQ ID NO:40 is the nucleotide sequence of the CT4R primer.

SEQ ID NO:41 is the nucleotide sequence of the CTSP1RecF primer.

SEQ ID NO:42 is the nucleotide sequence of the CTSP1RecR primer.

SEQ ID NO:43 is the nucleotide sequence of CTSP-1 exon 1.

SEQ ID NO:44 is the nucleotide sequence of CTSP-1 exon 2.

SEQ ID NO:45 is the nucleotide sequence of CTSP-1 exon 3.

SEQ ID NO:46 is the nucleotide sequence of CTSP-1 exon 4.

SEQ ID NO:47 is the nucleotide sequence of CTSP-1 exon 5.

SEQ ID NO:48 is the nucleotide sequence of CTSP-1 exon 6.

SEQ ID NO:49 is the nucleotide sequence of CTSP-1 exon 7.

SEQ ID NO:50 is the nucleotide sequence of CTSP-1 exon 8.

SEQ ID NO:51 is the nucleotide sequence of CTSP-1 exon 9 (contains a LTRrepeat).

SEQ ID NO:52 is the nucleotide sequence of CTSP-1 exon 10.

SEQ ID NO:53 is the nucleotide sequence of CTSP-1 exon 11.

SEQ 1D NO:54 is the nucleotide sequence of CTSP-1 exon 12 (contains anAlu repeat).

SEQ ID NO:55 is the nucleotide sequence of CTSP-1 exon 13.

SEQ ID NO:56 is the nucleotide sequence of CTSP-1 exon 14.

SEQ ID NO:57 is the nucleotide sequence of CTSP-1 exon 15.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to CTSP-1 antigen polypeptides provided herein andthe nucleic acid molecules that encode them, as well as related nucleicacid and polypeptide sequences corresponding to the related genesCTSP-2, CTSP-3 and CTSP-4. The invention further relates to the use ofthe nucleic acid molecules, polypeptides and fragments thereof inmethods and compositions for the diagnosis and treatment of cancer.

As used herein, the terms “CTSP nucleic acid”, “CTSP polypeptide” andthe like refer to a family of nucleic acids and polypeptides that are,or are related by a high degree of sequence identity to SEQ ID NOS: 1-8(nucleic acid sequence of CTSP-1 and splice variants thereof) or any ofSEQ ID NOS: 13-20 (polypeptide sequence of CTSP-1). Thus CTSP nucleicacids include SEQ ID NOs:1-11 (full length sequences), and partialsequences such as exons 1-15 of CTSP-1 (SEQ ID NOs:43-57) and exons ofrelated genes CTSP-2, CTSP-3 and CTSP-4. CTSP polypeptides include SEQID NO:13-23. The polypeptides elicit specific immune responses as isshown in the Examples below, and thus include CSTP polypeptides(including proteins) and fragments of CTSP polypeptides that arerecognized by the immune system (e.g., by antibodies and/or Tlymphocytes).

In part, the invention relates to CTSP polypeptides as well as thenucleic acid molecules that encode the CTSP polypeptides. As usedherein, the “nucleic acid molecules that encode” means the nucleic acidmolecules that code for the CTSP polypeptides or fragments thereof;particularly immunogenic fragments. These nucleic acid molecules may beDNA or may be RNA (e.g., mRNA). The CTSP nucleic acid molecules of theinvention also encompass variants of the nucleic acid moleculesdescribed herein. These variants may be splice variants, some of whichare described herein for CTSP-1, or allelic variants of certainsequences provided. Variants of the nucleic acid molecules of theinvention are intended to include homologs and alleles which aredescribed further below. Further, as used herein, the term “CTSPmolecules” includes CTSP polypeptides and fragments thereof as well asCTSP nucleic acids and fragments (such as exon sequences). In allembodiments, human CTSP polypeptides and the nucleic acid molecules thatencode them are preferred.

In one aspect, the invention provides isolated nucleic acid moleculesthat encode the CTSP polypeptides described herein. The isolated nucleicacid molecules of this aspect of the invention comprise: (a) nucleotidesequences set forth as SEQ ID NOs: 1-11 (b) isolated nucleic acidmolecules which hybridize under highly stringent conditions to thenucleic acid molecules of (a) and preferably which code for a CTSPpolypeptide, (c) nucleic acid molecules that differ from (a) or (b) dueto the degeneracy of the genetic code, and (d) full-length complementsof (a), (b) or (c).

As used herein the term “isolated nucleic acid molecule” means: (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a small percentage ofthe material in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

The CTSP nucleic acid molecules of the invention also encompass homologsand alleles which can be identified by conventional techniques.Identification of human and other organisms' homologs of CTSPpolypeptides will be familiar to those of skill in the art. In general,nucleic acid hybridization is a suitable method for identification ofhomologous sequences of another species (e.g., human, cow, sheep, dog,rat, mouse), which correspond to a known sequence. Standard nucleic acidhybridization procedures can be used to identify related nucleic acidsequences of selected percent identity. For example, one can construct alibrary of cDNAs reverse transcribed from the mRNA of a selected tissueand use the CTSP nucleic acid molecules identified herein to screen thelibrary for related nucleotide sequences. The screening preferably isperformed using high-stringency conditions to identify those sequencesthat are closely related by sequence identity. Nucleic acids soidentified can be translated into polypeptides and the polypeptides canbe tested for activity.

The term “high stringency” as used herein refers to parameters withwhich the art is familiar. Nucleic acid hybridization parameters may befound in references that compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, high-stringencyconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄ (pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDSis sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed, for example, in 2×SSC at room temperature and then at0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C.

There are other conditions, reagents, and so forth that can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of the CTSP nucleic acids of the invention(e.g., by using lower stringency conditions). The skilled artisan alsois familiar with the methodology for screening cells and libraries forexpression of such molecules, which then are routinely isolated,followed by isolation of the pertinent nucleic acid molecule andsequencing.

In general, homologs and alleles typically will share at least 90%nucleotide identity and/or amino acid identity to the sequences of CTSPnucleic acids and polypeptides, respectively, in some instances willshare at least 95% nucleotide identity and/or amino acid identity, inother instances will share at least 97% nucleotide identity and/or aminoacid identity, in other instances will share at least 98% nucleotideidentity and/or amino acid identity, and in other instances will shareat least 99% nucleotide identity and/or amino acid identity. Thehomology can be calculated using various, publicly available softwaretools developed by NCBI (Bethesda, Md.) that can be obtained through theinternet. Exemplary tools include the BLAST system available from thewebsite of the National Center for Biotechnology Information (NCBI) atthe National Institutes of Health. Pairwise and ClustalW alignments(BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysiscan be obtained using a number of sequence analysis software programs,such as the MacVector sequence analysis software (Accelrys SoftwareInc., San Diego, Calif.). Watson-Crick complements of the foregoingnucleic acids also are embraced by the invention.

In another aspect of the invention, unique fragments are provided whichinclude unique fragments of the nucleotide sequences of the inventionand complements thereof. The invention, in a preferred embodiment,provides unique fragments of SEQ ID NO:1-11 and 43-57 and complementsthereof. A unique fragment is one that is a ‘signature’ for the largernucleic acid. It, for example, is long enough to assure that its precisesequence is not found in molecules outside of the nucleic acid moleculesthat encode the CTSP polypeptides defined above. Those of ordinary skillin the art may apply no more than routine procedures to determine if afragment is unique within the human genome. In some instances the uniquefragment is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100 amino acids inlength.

Unique fragments can be used as probes in Southern blot assays toidentify such nucleic acid molecules, or can be used as probes inamplification assays such as those employing the polymerase chainreaction (PCR), including, but not limited to RT-PCR and RT-real-timePCR. As known to those skilled in the art, large probes such as 200nucleotides or more are preferred for certain uses such as Southernblots, while smaller fragments will be preferred for uses such as PCR.Unique fragments also can be used to produce fusion proteins forgenerating antibodies or determining binding of the polypeptidefragments, or for generating immunoassay components. Likewise, uniquefragments can be employed to produce nonfused fragments of the CTSPpolypeptides useful, for example, in the preparation of antibodies andin immunoassays.

In screening for CTSP genes, a Southern blot may be performed using theforegoing conditions, together with a detectably labeled probe (e.g.,radioactive or chemiluminescent probes). After washing the membrane towhich the DNA is finally transferred, the membrane can be placed againstX-ray film or analyzed using a phosphorimager device to detect theradioactive or chemiluminescent signal. In screening for the expressionof CTSP nucleic acids, Northern blot hybridizations using the foregoingconditions can be performed on samples taken from cancer patients orsubjects suspected of having a condition characterized by abnormal cellproliferation or neoplasia. Amplification protocols such as polymerasechain reaction using primers that hybridize to the sequences presentedalso can be used for detection of the CTSP genes or expression thereof.

Identification of related sequences can also be achieved usingpolymerase chain reaction (PCR) and other amplification techniquessuitable for cloning related nucleic acid sequences. Preferably, PCRprimers are selected to amplify portions of a nucleic acid sequencebelieved to be conserved (e.g., a catalytic domain, a DNA-bindingdomain, etc.). Again, nucleic acids are preferably amplified from atissue-specific library (e.g., testis). One also can use expressioncloning utilizing the antisera described herein to identify nucleicacids that encode related antigenic proteins in humans or other speciesusing the SEREX procedure to screen the appropriate expressionlibraries. (See: Sahin et al., 1995, Proc. Natl. Acad. Sci. U.S.A.92:11810-11813).

The invention also includes degenerate nucleic acids that includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating CTSPpolypeptide. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to: CCA, CCC, CCG, andCCT (proline codons); CGA, CGC, CGG, CGT, AGA, and AGG (argininecodons); ACA, ACC, ACG, and ACT (threonine codons); AAC and AAT(asparagine codons); and ATA, ATC, and ATT (isoleucine codons). Otheramino acid residues may be encoded similarly by multiple nucleotidesequences. Thus, the invention embraces degenerate nucleic acids thatdiffer from the biologically isolated nucleic acids in codon sequencedue to the degeneracy of the genetic code.

The invention also provides modified nucleic acid molecules, whichinclude additions, substitutions and deletions of one or morenucleotides (preferably 1-20 nucleotides). In preferred embodiments,these modified nucleic acid molecules and/or the polypeptides theyencode retain at least one activity or function of the unmodifiednucleic acid molecule and/or the polypeptides, such as antigenicity,receptor binding, etc. In certain embodiments, the modified nucleic acidmolecules encode modified polypeptides, preferably polypeptides havingconservative amino acid substitutions as are described elsewhere herein.The modified nucleic acid molecules are structurally related to theunmodified nucleic acid molecules and in preferred embodiments aresufficiently structurally related to the unmodified nucleic acidmolecules so that the modified and unmodified nucleic acid moleculeshybridize under stringent conditions known to one of skill in the art.

For example, modified nucleic acid molecules that encode polypeptideshaving single amino acid changes can be prepared. Each of these nucleicacid molecules can have one, two or three nucleotide substitutionsexclusive of nucleotide changes corresponding to the degeneracy of thegenetic code as described herein. Likewise, modified nucleic acidmolecules that encode polypeptides having two amino acid changes can beprepared which have, e.g., 2-6 nucleotide changes. Numerous modifiednucleic acid molecules like these will be readily envisioned by one ofskill in the art, including for example, substitutions of nucleotides incodons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and soon.

In the foregoing example, each combination of two amino acids isincluded in the set of modified nucleic acid molecules, as well as allnucleotide substitutions which code for the amino acid substitutions.Additional nucleic acid molecules that encode polypeptides havingadditional substitutions (i.e., 3 or more), additions or deletions(e.g., by introduction of a stop codon or a splice site(s)) also can beprepared and are embraced by the invention as readily envisioned by oneof ordinary skill in the art. Any of the foregoing nucleic acids orpolypeptides can be tested by routine experimentation for retention ofactivity or structural relation to the nucleic acids and/or polypeptidesdisclosed herein. As used herein the terms: “deletion”, “addition”, and“substitution” mean deletion, addition, and substitution changes toabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleic acids of asequence of the invention.

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the invention,preferably operably linked to a promoter is provided. In a relatedaspect, host cells transformed or transfected with such expressionvectors also are provided. As used herein, a “vector” may be any of anumber of nucleic acid molecules into which a desired sequence may beinserted by restriction and ligation for transport between differentgenetic environments or for expression in a host cell. Vectors aretypically composed of DNA although RNA vectors are also available.Vectors include, but are not limited to, plasmids, phagemids, and virusgenomes. A cloning vector is one which is able to replicate in a hostcell, and which is further characterized by one or more endonucleaserestriction sites at which the vector may be cut in a determinablefashion and into which a desired DNA sequence may be ligated such thatthe new recombinant vector retains its ability to replicate in the hostcell. In the case of plasmids, replication of the desired sequence mayoccur many times as the plasmid increases in copy number within the hostbacterium or just a single time per host before the host reproduces bymitosis. In the case of phage, replication may occur actively during alytic phase or passively during a lysogenic phase. An expression vectoris one into which a desired DNA sequence may be inserted by restrictionand ligation such that it is operably joined to regulatory sequences andmay be expressed as an RNA transcript. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellswhich have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art, e.g., β-galactosidase or alkalinephosphatase, and genes which visibly affect the phenotype of transformedor transfected cells, hosts, colonies or plaques, e.g., greenfluorescent protein. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. As used herein,“operably joined” and “operably linked” are used interchangeably andshould be construed to have the same meaning. If it is desired that thecoding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region is operably joined to a coding sequence if thepromoter region is capable of effecting transcription of that DNAsequence such that the resulting transcript can be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Often, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

It will also be recognized that the invention embraces the use of theCTSP nucleic acid molecules and genomic sequences in expression vectors,as well as to transfect host cells and cell lines, be these prokaryotic,e.g., E. coli, or eukaryotic, e.g., CHO cells, COS cells, yeastexpression systems, and recombinant baculovirus expression in insectcells. Especially useful are mammalian cells such as human, mouse,hamster, pig, goat, primate, etc. They may be of a wide variety oftissue types, including mast cells, fibroblasts, oocytes, andlymphocytes, and may be primary cells and cell lines. Specific examplesinclude dendritic cells, peripheral blood leukocytes, bone marrow stemcells and embryonic stem cells. The expression vectors require that thepertinent sequence, i.e., those nucleic acids described supra, beoperably linked to a promoter.

The invention, in one aspect, also permits the construction of CTSP gene“knock-outs” and “knock-ins” in cells and in animals, providingmaterials for studying certain aspects of cancer and immune systemresponses to cancer.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. Cellsare genetically engineered by the introduction into the cells ofheterologous DNA or RNA encoding a CTSP polypeptide, a mutant CTSPpolypeptide, fragments, or variants thereof. The heterologous DNA or RNAis placed under operable control of transcriptional elements to permitthe expression of the heterologous DNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pcDNA3.1 and pCDM8 (Invitrogen) that contain a selectable marker(which facilitates the selection of stably transfected cell lines) andcontain the human cytomegalovirus (CMV) enhancer-promoter sequences.Additionally, suitable for expression in primate or canine cell lines isthe pCEP4 vector (Invitrogen), which contains an Epstein Barr virus(EBV) origin of replication, facilitating the maintenance of plasmid asa multicopy extrachromosomal element. Another expression vector is thepEF-BOS plasmid containing the promoter of polypeptide Elongation Factor1, which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mizushima and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdescribed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996).

The invention also embraces kits termed expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also includes kits for amplification of a CTSP nucleicacid, including at least one pair of amplification primers whichhybridize to a CTSP nucleic acid. The primers preferably are about 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31 or 32 nucleotides in length and are non-overlapping to preventformation of “primer-dimers”. One of the primers will hybridize to onestrand of the CTSP nucleic acid and the second primer will hybridize tothe complementary strand of the CTSP nucleic acid, in an arrangementthat permits amplification of the CTSP nucleic acid. Selection ofappropriate primer pairs is standard in the art. For example, theselection can be made with assistance of a computer program designed forsuch a purpose, optionally followed by testing the primers foramplification specificity and efficiency.

The invention, in another aspect provides isolated polypeptides(including whole proteins and partial proteins) encoded by the foregoingCTSP nucleic acids. Examples of the amino acid sequences encoded by theforegoing CTSP nucleic acids are set forth as any of SEQ ID NOs: 13-23.The amino acids of the invention are also intended to encompass aminoacid sequences that result from the translation of the nucleic acidsequences provided herein in a different reading frame. In one preferredembodiment of the invention a polypeptide is provided which comprisesthe polypeptide sequence set forth as any of SEQ ID NOS: 13-20.

Such polypeptides are useful, for example, alone or as fusion proteinsto generate antibodies, and as components of an immunoassay ordiagnostic assay. Immunogenic CTSP polypeptides can be isolated frombiological samples including tissue or cell homogenates, and can also beexpressed recombinantly in a variety of prokaryotic and eukaryoticexpression systems by constructing an expression vector appropriate tothe expression system, introducing the expression vector into theexpression system, and isolating the recombinantly expressed protein.Fragments of the immunogenic CTSP polypeptides (including immunogenicpeptides) also can be synthesized chemically using well-establishedmethods of peptide synthesis. Thus, fragments of the disclosedpolypeptides are useful for eliciting an immune response. In oneembodiment fragments of a polypeptide which comprises any of SEQ ID NO:13-23 that are at least eight amino acids in length and exhibitimmunogenicity are provided. In one embodiment fragments of apolypeptide which comprises any of SEQ ID NOS: 13-20 that are at leasteight amino acids in length and exhibit immunogenicity are provided.

Fragments of a polypeptide preferably are those fragments that retain adistinct functional capability of the polypeptide. Functionalcapabilities that can be retained in a fragment of a polypeptide includeinteraction with antibodies or MHC molecules (e.g. immunogenicfragments), interaction with other polypeptides or fragments thereof,selective binding of nucleic acids or proteins, and enzymatic activity.One important activity is the ability to provoke in a subject an immuneresponse. As will be recognized by those skilled in the art, the size ofthe fragment that can be used for inducing an immune response willdepend upon factors such as whether the epitope recognized by anantibody is a linear epitope or a conformational epitope or theparticular MHC molecule that binds to and presents the fragment (e.g.,HLA class I or II). Thus, some immunogenic fragments of CTSPpolypeptides will consist of longer segments while others will consistof shorter segments, (e.g., about 5, 6, 7, 8, 9, 10, 11 or 12 or moreamino acids long, including each integer up to the full length of theCTSP polypeptide). Those skilled in the art are well versed in methodsfor selecting immunogenic fragments of polypeptides.

The invention embraces variants of the CTSP polypeptides describedabove. As used herein, a “variant” of a CTSP polypeptide is apolypeptide which contains one or more modifications to the primaryamino acid sequence of a CTSP polypeptide. Modifications which create aCTSP polypeptide variant can be made to a CTSP polypeptide 1) to reduceor eliminate an activity of a CTSP polypeptide; 2) to enhance a propertyof a CTSP polypeptide, such as protein stability in an expression systemor the stability of protein-protein binding; 3) to provide a novelactivity or property to a CTSP polypeptide, such as addition of anantigenic epitope or addition of a detectable moiety; or 4) to provideequivalent or better binding to a MHC molecule.

Modifications to a CTSP polypeptide are typically made to the nucleicacid which encodes the CTSP polypeptide, and can include deletions,point mutations, truncations, amino acid substitutions and additions ofamino acids or non-amino acid moieties. Alternatively, modifications canbe made directly to the polypeptide, such as by cleavage, addition of alinker molecule, addition of a detectable moiety, such as biotin,addition of a fatty acid, and the like. Modifications also embracefusion proteins comprising all or part of the CTSP polypeptide aminoacid sequence. One of skill in the art will be familiar with methods forpredicting the effect on protein conformation of a change in proteinsequence, and can thus “design” a variant CTSP polypeptide according toknown methods. One example of such a method is described by Dahiyat andMayo in Science 278:82-87, 1997, whereby proteins can be designed denovo. The method can be applied to a known protein to vary a only aportion of the polypeptide sequence. By applying the computationalmethods of Dahiyat and Mayo, specific variants of a CTSP polypeptide canbe proposed and tested to determine whether the variant retains adesired conformation.

In general, variants include CTSP polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsdesired physiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aCTSP polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a CTSP polypeptide preferablypreserve the amino acid reading frame of the coding sequence, andpreferably do not create regions in the nucleic acid which are likely tohybridize to form secondary structures, such a hairpins or loops, whichcan be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant CTSP polypeptides) which are silent as tothe amino acid sequence of the polypeptide, but which provide preferredcodons for translation in a particular host. The preferred codons fortranslation of a nucleic acid in, e.g., E. coli, are well known to thoseof ordinary skill in the art. Still other mutations can be made to thenoncoding sequences of a CTSP gene or cDNA clone to enhance expressionof the polypeptide. The activity of variants of CTSP polypeptides can betested by cloning the gene encoding the variant CTSP polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the variant CTSP polypeptide, andtesting for a functional capability of the CTSP polypeptides asdisclosed herein. For example, the variant CTSP polypeptide can betested for reaction with autologous or allogeneic sera as described inthe Examples. Preparation of other variant polypeptides may favortesting of other activities, as will be known to one of ordinary skillin the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in immunogenic CTSP polypeptides to providefunctionally equivalent variants, or homologs of the foregoingpolypeptides, i.e., the variants retain the functional capabilities ofthe immunogenic CTSP polypeptides. As used herein, a “conservative aminoacid substitution” refers to an amino acid substitution that does notalter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Variants can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references that compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York, 1989, or Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.Exemplary functionally equivalent variants or homologs of the CTSPpolypeptides include conservative amino acid substitutions of in theamino acid sequences of proteins disclosed herein. Conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, one canmake conservative amino acid substitutions to the amino acid sequence ofthe CTSP polypeptides disclosed herein and retain the specificantibody-binding characteristics of the antigens.

Likewise, upon determining that a peptide derived from a CTSPpolypeptide is presented by an MHC molecule and recognized by antibodiesor T lymphocytes (e.g., helper T cells or CTLs), one can makeconservative amino acid substitutions to the amino acid sequence of thepeptide, particularly at residues which are thought not to be directcontact points with the MHC molecule. For example, methods foridentifying functional variants of HLA class II binding peptides areprovided in a published PCT application of Strominger and Wucherpfennig(PCT/US96/03182). Peptides bearing one or more amino acid substitutionsalso can be tested for concordance with known HLA/MHC motifs prior tosynthesis using, e.g. the computer program described by D'Amaro andDrijfhout (D'Amaro et al., Human Immunol. 43:13-18, 1995; Drijfhout etal., Human Immunol. 43:1-12, 1995). The substituted peptides can then betested for binding to the MHC molecule and recognition by antibodies orT lymphocytes when bound to MHC. These variants can be tested forimproved stability and are useful, inter alfa, in vaccine compositions.

Conservative amino-acid substitutions in the amino acid sequence of CTSPpolypeptides to produce functionally equivalent variants of CTSPpolypeptides typically are made by alteration of a nucleic acid encodinga CTSP polypeptide. Such substitutions can be made by a variety ofmethods known to one of ordinary skill in the art. For example, aminoacid substitutions may be made by PCR-directed mutation, site-directedmutagenesis according to the method of Kunkel (Kunkel, 1985, Proc. Nat.Acad. Sci. U.S.A. 82: 488-492), or by chemical synthesis of a geneencoding a CTSP polypeptide. Where amino acid substitutions are made toa small unique fragment of a CTSP polypeptide, such as an antigenicepitope recognized by autologous or allogeneic sera or T lymphocytes,the substitutions can be made by directly synthesizing the peptide. Theactivity of functionally equivalent variants of CTSP polypeptides can betested by cloning the gene encoding the altered CTSP polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered polypeptide, and testingfor a functional capability of the CTSP polypeptides as disclosedherein. Peptides that are chemically synthesized can be tested directlyfor function, e.g., for binding to antisera recognizing associatedantigens.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. In one aspect of the invention a methodof identifying polypeptides homologous to CTSP is provided. Novel CTSPpolypeptides can be identified by obtaining a biological sample from asubject, determining the reactivity of the biological sample with one ormore known CTSP polypeptides (as described herein), and subsequentlyusing the reactive biological sample to screen an expression library toidentify novel CTSP polypeptides.

As used herein, a “subject” is preferably a human, non-human primate,cow, horse, pig, sheep, goat, dog, cat or rodent. In all embodiments,human subjects are preferred. In some embodiments, the subject issuspected of having cancer or has been diagnosed with cancer. Cancers inwhich the CTSP nucleic acid or polypeptide are differentially expressedinclude cancers of the breast, colon, esophagus, glioblastoma, lung,melanoma, prostate, stomach, thyroid, and uterus.

As used herein, a biological sample includes, but is not limited to:tissue, cells, and/or body fluid (e.g., serum, blood, lymph node fluid,etc.). The fluid sample may include cells and/or fluid. The tissue andcells may be obtained from a subject or may be grown in culture (e.g.,from a cell line). As used herein, a biological sample is body fluid,tissue or cells obtained from a subject using methods well-known tothose of ordinary skill in the related medical arts. Typically, abiological sample may be obtained by collecting a blood sample or abiopsy sample from a subject. The biological sample can include tumortissue or cells, normal tissue or cells, or combinations thereof.

The invention in another aspect permits the isolation of thecancer-associated antigens described herein. A variety of methodologieswell-known to the skilled practitioner can be utilized to obtainisolated cancer-associated antigens. The proteins may be purified fromcells which naturally produce the protein by chromatographic means orimmunological recognition. Alternatively, an expression vector may beintroduced into cells to cause production of the protein. In anothermethod, mRNA transcripts may be microinjected or otherwise introducedinto cells to cause production of the encoded protein. Translation ofmRNA in cell-free extracts such as the reticulocyte lysate system alsomay be used to produce the protein. Those skilled in the art also canreadily follow known methods for isolating cancer-associated antigens.These include, but are not limited to, chromatographic techniques suchas immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography, and immune-affinity chromatography.

The invention also involves diagnosing or monitoring cancer in subjectsby determining the presence of an immune response to one or more CTSPpolypeptides of the invention. In preferred embodiments, thisdetermination is performed by assaying a bodily fluid obtained from thesubject, preferably serum, blood, or lymph node fluid for the presenceof antibodies against the CTSP polypeptides described herein. Thisdetermination may also be performed by assaying a tissue or cells fromthe subject for the presence of one or more CTSP polypeptides (ornucleic acid molecules that encode these polypeptides) described herein.In another embodiment, the presence of antibodies against at least oneadditional cancer-associated antigen or cancer-testis antigen isdetermined for diagnosis of cancer. This determination may also beperformed by assaying a tissue or cells from the subject for thepresence of the CTSP polypeptides described herein.

Measurement of the expression of CTSP polypeptides or nucleic acidmolecules, or the immune response against one of the CTSP polypeptides,over time by sequential determinations permits monitoring of the diseaseand/or the effects of a course of treatment. For example, a sample, suchas serum, blood, or lymph node fluid, may be obtained from a subject,tested for expression of CTSP molecules or an immune response to one ofthe CTSP polypeptides, and at a second, subsequent time, another sample,may be obtained from the subject and similarly tested. The results ofthe first and second (or subsequent) tests can be compared as a measureof the onset, regression or progression of cancer, or, if cancertreatment was undertaken during the interval between obtaining thesamples, the effectiveness of the treatment may be evaluated bycomparing the results of the two tests. In preferred embodiments ofimmune response testing, the CTSP polypeptides are bound to a substrateand/or the immune response to the CTSP polypeptides is determined withELISA. Other methods will be apparent to one of skill in the art.

Diagnostic methods of the invention also involve determining theaberrant expression of one or more of the CTSP polypeptides describedherein or the nucleic acid molecules that encode them. Suchdeterminations can be carried out via any standard nucleic acid assay,including the polymerase chain reaction or assaying with hybridizationprobes, which may be labeled, or by assaying biological samples withbinding partners (e.g., antibodies) for CTSP polypeptides.

The diagnostic methods of the invention can be used to detect thepresence of a disorder associated with aberrant expression of a CTSPmolecule (e.g., onset of the disorder), as well as to assess theprogression and/or regression of the disorder such as in response totreatment (e.g., chemotherapy, radiation). According to this aspect ofthe invention, the method for diagnosing a disorder characterized byaberrant expression of a CTSP molecule involve: detecting expression ofa CTSP molecule in a first biological sample obtained from a subject,wherein differential expression of the CTSP molecule compared to acontrol sample indicates that the subject has a disorder characterizedby aberrant expression of a CTSP molecule, such as cancer.

As described herein, CTSP molecules are expressed in testis tissue andcertain other normal tissues (brain, fetal brain and spinal cord forCTSP-2). Therefore, in all of the diagnostic methods described herein,the biological sample preferably does not contain testis cells or testistissue in order to avoid false-positive results. For CTSP-2, the samplepreferably also does not contain brain, fetal brain or spinal cord cellsor tissue.

As used herein, “aberrant expression” of a CTSP molecule is intended toinclude any expression that is statistically significant different fromthe expected (e.g., normal or baseline) amount of expression. Forexample, expression of a CTSP molecule (i.e., CTSP polypeptides or thenucleic acid molecules that encode them) in a tissue that is notexpected to express the CTSP molecule would be included in thedefinition of “aberrant expression”. Likewise, expression of the CTSPmolecule that is determined to be expressed at a significantly higher orlower level than expected is also included. Therefore, a determinationof the level of expression of one or more of the CTSP polypeptidesand/or the nucleic acids that encode them is diagnostic of cancer if thelevel of expression is above a baseline level determined for that tissuetype. The baseline level of expression can be determined using standardmethods known to those of skill in the art. Such methods include, forexample, assaying a number of histologically normal tissue samples(preferably not testis) from subjects that are clinically normal (i.e.do not have clinical signs of cancer in that tissue type) anddetermining the mean level of expression for the samples.

The level of expression of the nucleic acid molecules of the inventionor the polypeptides they encode can indicate cancer in the tissue whenthe level of expression is significantly more in the tissue than in acontrol sample. In some embodiments, a level of expression in thetissues that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, or 500% more than thelevel of expression in the control tissue indicates cancer in thetissue.

As used herein the term “control” means predetermined values, and alsomeans samples of materials tested in parallel with the experimentalmaterials. Examples include samples from control populations, biopsysamples taken from tissue adjacent to a biopsy sample suspected of beingcancerous and control samples generated through manufacture to be testedin parallel with the experimental samples.

As used herein the term “control” includes positive and negativecontrols which may be a predetermined value that can take a variety offorms. The control(s) can be a single cut-off value, such as a median ormean, or can be established based upon comparative groups, such as ingroups having normal amounts of CTSP molecules of the invention andgroups having abnormal amounts of CTSP molecules of the invention.Another example of a comparative group is a group having a particulardisease, condition and/or symptoms and a group without the disease,condition and/or symptoms. Another comparative group is a group with afamily history of a particular disease and a group without such a familyhistory of the particular disease. The predetermined control value canbe arranged, for example, where a tested population is divided equally(or unequally) into groups, such as a low-risk group, a medium-riskgroup and a high-risk group or into quadrants or quintiles, the lowestquadrant or quintile being individuals with the lowest risk or lowestexpression levels of a CTSP molecule of the invention that isup-regulated in cancer and the highest quadrant or quintile beingindividuals with the highest risk or highest expression levels of a CTSPmolecule of the invention that is up-regulated in cancer.

The predetermined value of a control will depend upon the particularpopulation selected. For example, an apparently healthy population willhave a different “normal” CTSP molecule expression level range than willa population which is known to have a condition characterized byaberrant expression of the CTSP molecule. Accordingly, the predeterminedvalue selected may take into account the category in which an individualfalls. Appropriate ranges and categories can be selected with no morethan routine experimentation by those of ordinary skill in the art.Typically the control will be based on apparently healthy individuals inan appropriate age bracket. As used herein, the term “increasedexpression” means a higher level of expression relative to a selectedcontrol.

The invention involves in some aspects diagnosing or monitoring cancerby determining the level of expression of one or more CTSP nucleic acidmolecules and/or determining the level of expression of one or more CTSPpolypeptides they encode. In some important embodiments, thisdetermination is performed by assaying a tissue sample from a subjectfor the level of expression of one or more CTSP nucleic acid moleculesor for the level of expression of one or more CTSP polypeptides encodedby the nucleic acid molecules of the invention.

The expression of the molecules of the invention may be determined usingroutine methods known to those of ordinary skill in the art. Thesemethods include, but are not limited to: direct RNA amplification,reverse transcription of RNA to cDNA, real-time RT-PCR, amplification ofcDNA, hybridization, and immunologically based assay methods, whichinclude, but are not limited to immunohistochemistry, antibody sandwichcapture assay, ELISA, and enzyme-linked immunospot assay (EliSpotassay). For example, the determination of the presence of level ofnucleic acid molecules of the invention in a subject or tissue can becarried out via any standard nucleic acid determination assay, includingthe) polymerase chain reaction, or assaying with labeled hybridizationprobes. Such hybridization methods include, but are not limited tomicroarray techniques.

These methods of determining the presence and/or level of the moleculesof the invention in cells and tissues may include use of labels tomonitor the presence of the molecules of the invention. Such labels mayinclude, but are not limited to, radiolabels or chemiluminescent labels,which may be utilized to determine whether a molecule of the inventionis expressed in a cell or tissue, and to determine the level ofexpression in the cell or tissue. For example, a fluorescently labeledor radiolabeled antibody that selectively binds to a polypeptide of theinvention may be contacted with a tissue or cell to visualize thepolypeptide in vitro or in vivo. These and other in vitro and in vivoimaging methods for determining the presence of the nucleic acid andpolypeptide molecules of the invention are well known to those ofordinary skill in the art.

The invention, therefore, also involves the use of agents such aspolypeptides that bind to CTSP polypeptides. Such agents can be used inmethods of the invention including the diagnosis and/or treatment ofcancer. Such binding agents can be used, for example, in screeningassays to detect the presence or absence of CTSP polypeptides and can beused in quantitative binding assays to determine levels of expression inbiological samples and cells. Such agents also may, be used to inhibitthe native activity of the CTSP polypeptides, for example, by binding tosuch polypeptides.

According to this aspect, the binding polypeptides bind to an isolatednucleic acid or protein of the invention, including unique fragmentsthereof. Preferably, the binding polypeptides bind to a CTSPpolypeptide, or a unique fragment thereof.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, more preferably, an Fab or F(ab)₂ fragment of anantibody. Typically, the fragment includes a CDR3 region that isselective for the CTSP polypeptide. Any of the various types ofantibodies can be used for this purpose, including polyclonalantibodies, monoclonal antibodies, humanized antibodies, and chimericantibodies.

Thus, the invention provides agents which bind to CTSP polypeptidesencoded by CTSP nucleic acid molecules of the invention, and in certainembodiments preferably to unique fragments of the CTSP polypeptides.Such binding partners can be used in screening assays to detect thepresence or absence of a CTSP polypeptide and in purification protocolsto isolate such CTSP polypeptides. Likewise, such binding partners canbe used to selectively target drugs, toxins or other molecules(including detectable diagnostic molecules) to cells which express CTSPpolypeptides. In this manner, for example, cells present in solid ornon-solid tumors which express CTSP polypeptides can be treated withcytotoxic compounds that are selective for the CTSP molecules (nucleicacids and/or antigens). Such binding agents also can be used to inhibitthe native activity of the CTSP polypeptide, for example, to furthercharacterize the functions of these molecules.

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering a protein, fragments of aprotein, cells expressing the protein or fragments thereof and the liketo an animal to induce polyclonal antibodies. The present invention alsoprovides methods of producing monoclonal antibodies to the CTSPmolecules of the invention described herein. The production ofmonoclonal antibodies is performed according to techniques well known inthe art. As detailed herein, such antibodies may be used for example toidentify tissues expressing protein or to purify protein. Antibodiesalso may be coupled to specific labeling agents or imaging agents,including, but not limited to a molecule preferably selected from thegroup consisting of fluorescent, enzyme, radioactive, metallic, biotin,chemiluminescent, bioluminescent, chromophore, or colored, etc. In someaspects of the invention, a label may be a combination of the foregoingmolecule types.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R., 1986, TheExperimental Foundations of Modern Immunology, Wiley & Sons, Inc., NewYork; Roitt, I., 1991, Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)2 fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Fab fragments consist of acovalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd. The Fd fragments are the major determinant ofantibody specificity (a single Fd fragment may be associated with up toten different light chains without altering antibody specificity) and Fdfragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762, and 5,859,205.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. Following immunization of these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (HAMA) responseswhen administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)2, Fab, Fv, and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)2 fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies, domain antibodies and heavy chain antibodies(Ablynx NV, Ghent, Belgium).

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to CTSP polypeptides. These polypeptides may bederived also from sources other than antibody technology. For example,such polypeptide binding agents can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilized formor as phage display libraries. Combinatorial libraries also can besynthesized of peptides containing one or more amino acids. Librariesfurther can be synthesized of peptides and non-peptide syntheticmoieties.

The CTSP polypeptides of the invention can be used to screen peptidelibraries, including phage display libraries, to identify and selectpeptide binding partners of the CTSP molecules of the invention. Suchmolecules can be used, as described, for screening assays, fordiagnostic assays, for purification protocols or for targeting drugs,toxins and/or labeling agents (e.g., radioisotopes, fluorescentmolecules, etc.) to cells which express CTSP molecules such as cancercells which have aberrant CTSP expression.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g., m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe CTSP polypeptide. This process can be repeated through severalcycles of reselection of phage that bind to the CTSP polypeptide.Repeated rounds lead to enrichment of phage bearing particularsequences. DNA sequence analysis can be conducted to identify thesequences of the expressed polypeptides. The minimal linear portion ofthe sequence that binds to the CTSP polypeptide can be determined. Onecan repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Yeasttwo-hybrid screening methods also may be used to identify polypeptidesthat bind to the CTSP polypeptides.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used to identify tissues with normal or aberrant expression of aCTSP polypeptide. Antibodies also may be coupled to specific diagnosticlabeling agents for imaging of cells and tissues with normal or aberrantCTSP polypeptide expression or to therapeutically useful agentsaccording to standard coupling procedures. As used herein,“therapeutically useful agents” include any therapeutic molecule whichdesirably is targeted selectively to a cell or tissue selectively withan aberrant CTSP expression.

Diagnostic agents for in vivo use include, but are not limited to,barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium,diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoatesodium and radiodiagnostics including positron emitters such asfluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99, iodine-131 and indium-111, and nuclides for nuclearmagnetic resonance such as fluorine and gadolinium. Other diagnosticagents useful in the invention will be apparent to one of ordinary skillin the art.

The antibodies of the present invention can also be used totherapeutically target CTSP polypeptides. In one embodiment, antibodiescan be used to target CTSP antigens expressed on the cell surface, suchas CTSP peptides presented by MHC molecules. This can be accomplished,for example, by raising antibodies that recognize the complex of CTSPpeptides and MHC molecules.

These antibodies can be linked not only to a detectable marker but alsoan antitumor agent or an immunomodulator. Antitumor agents can includecytotoxic agents and agents that act on tumor neovasculature. Detectablemarkers include, for example, radioactive or fluorescent markers.Cytotoxic agents include cytotoxic radionuclides, chemical toxins andprotein toxins.

The cytotoxic radionuclide or radiotherapeutic isotope preferably is analpha-emitting isotope such as ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁴Raor ²²³Ra. Alternatively, the cytotoxic radionuclide may a beta-emittingisotope such as ¹⁸⁶Rh, ¹⁸⁸ Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ⁶⁴Cu, ¹⁵³Sm or¹⁶⁶Ho. Further, the cytotoxic radionuclide may emit Auger and low energyelectrons and include the isotopes ¹²⁵I, ¹²³I or ⁷⁷Br.

Suitable chemical toxins or chemotherapeutic agents include members ofthe enediyne family of molecules, such as calicheamicin and esperamicin.Chemical toxins can also be taken from the group consisting ofmethotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine,mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.Other antineoplastic agents that may be conjugated to the antibodies ofthe present invention include dolastatins (U.S. Pat. Nos. 6,034,065 and6,239,104) and derivatives thereof. Of particular interest is dolastatin10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine) and thederivatives auristatin PHE(dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester)(Pettit, G. R. et al., 1998, Anticancer Drug Des. 13(4):243-277; Woyke,T. et al., 2001, Antimicrob. Agents Chemother. 45(12):3580-3584), andaurastatin E and the like. Toxins that are less preferred in thecompositions and methods of the invention include poisonous lectins,plant toxins such as ricin, abrin, modeccin, botulinum and diphtheriatoxins. Of course, combinations of the various toxins could also becoupled to one antibody molecule thereby accommodating variablecytotoxicity. Other chemotherapeutic agents are known to those skilledin the art.

Agents that act on the tumor vasculature can include tubulin-bindingagents such as combrestatin A4 (Griggs et al., 2001, Lancet Oncol.2:82), angiostatin and endostatin (reviewed in Rosen, 2000, Oncologist5:20, incorporated by reference herein) and interferon inducible protein10 (U.S. Pat. No. 5,994,292). A number of antiangiogenic agentscurrently in clinical trials are also contemplated. Agents currently inclinical trials include: 2ME2, Angiostatin, Angiozyme, Anti-VEGF RhuMAb,Apra (CT-2584), Avicine, Benefin, BMS275291, Carboxyamidotriazole,CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC 412), CM101,Combretastatin A-4 Prodrug, EMD 121974, Endostatin, Flavopiridol,Genistein (GCP), Green Tea Extract, IM-862, ImmTher, Interferon alpha,Interleukin-12, Iressa (ZD1839), Marimastat, Metastat (Col-3),Neovastat, Octreotide, Paclitaxel, Penicillamine, Photofrin, Photopoint,PI-88, Prinomastat (AG-3340), PTK787 (ZK22584), RO317453, Solimastat,Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE 26644), Suramin(Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 and Vitaxin.Additional antiangiogenic agents are described by Kerbel, 2001, J. Clin.Oncol. 19(18s):45s-51s, which is incorporated by reference herein.Immunomodulators suitable for conjugation to the antibodies includeα-interferon, γ-interferon, and tumor necrosis factor alpha (TNFα).

The coupling of one or more toxin molecules to the antibody isenvisioned to include many chemical mechanisms, for instance covalentbinding, affinity binding, intercalation, coordinate binding, andcomplexation. The toxic compounds used to prepare the immunotoxins areattached to the antibodies or antigen-binding fragments thereof bystandard protocols known in the art.

In other aspects of the invention, the CTSP molecules and the antibodiesand other binding molecules, as described herein, can be used for thetreatment of disorders. When “disorder” is used herein, it refers to anypathological condition where the CTSP polypeptides are aberrantlyexpressed. An example of such a disorder is cancer, with breast cancer,lung cancer, colon cancer, prostate cancer, esophageal cancer, braincancers such as glioblastoma, melanoma, stomach cancer, thyroid cancer,uterine cancer, ovarian cancer, renal cancer, sarcoma, leukemia,lymphoma, gastric cancer, glioma, bladder cancer, and hepatoma.

Conventional treatment for cancer may include, but is not limited to:surgical intervention, chemotherapy, radiotherapy, and adjuvant systemictherapies. In one aspect of the invention, treatment may includeadministering binding polypeptides such as antibodies that specificallybind to the CTSP polypeptide. These binding polypeptides can beoptionally linked to one or more detectable markers, antitumor agents orimmunomodulators as described above.

Cancer treatment, in another aspect of the invention may includeadministering antisense molecules or RNAi molecules to reduce expressionlevel and/or function level of CTSP polypeptides of the invention in thesubject in cancers where a CTSP molecule is up-regulated. The use of RNAinterference or “RNAi” involves the use of double-stranded RNA (dsRNA)to block gene expression. (see: Sui G et al., 2002, Proc Natl. Acad. SciU.S.A. 99:5515-5520). Methods of applying RNAi strategies in embodimentsof the invention would be understood by one of ordinary skill in theart.

CTSP polypeptides as described herein, can also be used in one aspect ofthe invention to induce or enhance an immune response. For example, theCTSP polypeptides of the invention may be used to stimulate lymphocytes,eliciting a B cell response and/or T cell response. Non-limitingexamples of T cell responses include CD4+ T cell responses and CD8+ cellresponses. Some therapeutic approaches based upon the disclosure arepremised on a response by a subject's immune system, leading to lysis ofantigen presenting cells, such as cancer cells which present one or moreCTSP polypeptides of the invention. One such approach is theadministration of autologous CTLs specific to a CTSP polypeptide/MHCcomplex to a subject with abnormal cells of the phenotype at issue. Itis within the ability of one of ordinary skill in the art to developsuch CTLs in vitro. An example of a method for T cell differentiation ispresented in International Application number PCT/US96/05607. Generally,a sample of cells taken from a subject, such as blood cells, arecontacted with a cell presenting the complex and capable of provokingCTLs to proliferate. The target cell can be a transfectant, such as aCOS cell. These transfectants present the desired complex of theirsurface and, when combined with a CTL of interest, stimulate itsproliferation. COS cells are widely available, as are other suitablehost cells. Specific production of CTL clones is well known in the art.The clonally expanded autologous CTLs then are administered to thesubject.

Another method for selecting antigen-specific CTL clones has recentlybeen described (Altman et al., 1996, Science 274:94-96; Dunbar et al.,1998, Curr. Biol. 8:413-416), in which fluorogenic tetramers of MHCclass I molecule/peptide complexes are used to detect specific CTLclones. Briefly, soluble MHC class I molecules are folded in vitro inthe presence of β₂-microglobulin and a peptide antigen which binds theclass I molecule. After purification, the MHC/peptide complex ispurified and labeled with biotin. Tetramers are formed by mixing thebiotinylated peptide-MHC complex with labeled avidin (e.g.,phycoerythrin) at a molar ratio or 4:1. Tetramers are then contactedwith a source of CTLs such as peripheral blood or lymph node. Thetetramers bind CTLs which recognize the peptide antigen/MHC class Icomplex. Cells bound by the tetramers can be sorted by fluorescenceactivated cell sorting to isolate the reactive CTLs. The isolated CTLsthen can be expanded in vitro for use as described herein.

To detail a therapeutic methodology, referred to as adoptive transfer(Greenberg, 1986, J. Immunol. 136(5): 1917; Riddel et al., 1992, Science257: 238; Lynch et al., 1991, Eur. J. Immunol. 21: 1403-1410; Kast etal., 1989, Cell 59: 603-614), cells presenting the desired complex(e.g., dendritic cells) are combined with CTLs leading to proliferationof the CTLs specific thereto. The proliferated CTLs are thenadministered to a subject with a cellular abnormality which ischaracterized by certain of the abnormal cells presenting the particularcomplex. The CTLs then lyse the abnormal cells, thereby achieving thedesired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/cancer associated antigencomplex. This can be determined very easily, as the art is very familiarwith methods for identifying cells which present a particular HLAmolecule, as well as how to identify cells expressing DNA of thepertinent sequences, in this case a CTSP polypeptide sequence. Oncecells presenting the relevant complex are identified via the foregoingscreening methodology, they can be combined with a sample from apatient, where the sample contains CTLs. If the complex presenting cellsare lysed by the mixed CTL sample, then it can be assumed that a CTSPpolypeptide is being presented, and the subject is an appropriatecandidate for the therapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach is the use of non-proliferativecells expressing the complex. The cells used in this approach may bethose that normally express the complex, such as irradiated tumor cellsor cells transfected with one or both of the genes necessary forpresentation of the complex (i.e., the antigenic peptide and thepresenting MHC molecule). Chen et al. (Proc. Natl. Acad. Sci. U.S.A. 88:110-114, 1991) exemplifies this approach, showing the use of transfectedcells expressing HPV E7 peptides in a therapeutic regime. Various celltypes may be used. Similarly, vectors carrying one or both of the genesof interest may be used. Viral or bacterial vectors are especiallypreferred. For example, nucleic acids which encode a CTSP polypeptidemay be operably linked to promoter and enhancer sequences which directexpression of the CTSP polypeptide in certain tissues or cell types. Thenucleic acid may be incorporated into an expression vector.

Expression vectors may be unmodified extrachromosomal nucleic acids,plasmids or viral genomes constructed or modified to enable insertion ofexogenous nucleic acids, such as those encoding CTSP polypeptide, asdescribed elsewhere herein. Nucleic acids encoding a CTSP polypeptidealso may be inserted into a retroviral genome, thereby facilitatingintegration of the nucleic acid into the genome of the target tissue orcell type. In these systems, the gene of interest is carried by amicroorganism, e.g., a Vaccinia virus, pox virus, herpes simplex virus,retrovirus or adenovirus, and the materials de facto “infect” hostcells. The cells which result present the complex of interest, and arerecognized by autologous CTLs, which then proliferate.

A similar effect can be achieved by combining the CTSP polypeptide or astimulatory fragment thereof with an adjuvant to facilitateincorporation into antigen presenting cells in vivo. The CTSPpolypeptide is processed to yield the peptide partner of the MHCmolecule while a CTSP fragment may be presented without the need forfurther processing. Generally, subjects can receive an intradermalinjection of an effective amount of the CTSP polypeptide. Initial dosescan be followed by booster doses, following immunization protocolsstandard in the art. Preferred CTSP polypeptides include those found toreact with allogeneic cancer antisera, shown in the examples below.

The invention involves the use of various materials disclosed herein to“immunize” subjects or as “vaccines”. As used herein, “immunization” or“vaccination” means increasing or activating an immune response againstan antigen. It does not require elimination or eradication of acondition but rather contemplates the clinically favorable enhancementof an immune response toward an antigen. Generally accepted animalmodels, can be used for testing of immunization against cancer using aCTSP molecule. For example, human cancer cells can be introduced into amouse to create a tumor, and one or more CTSP polypeptides or fragmentsthereof can be delivered, optionally combined with one or more adjuvantsand/or cytokines to boost the immune response. The effect on the cancercells (e.g., reduction of tumor size) can be assessed as a measure ofthe effectiveness of the CTSP immunization. Testing of the foregoinganimal model using other methods for immunization include theadministration of one or more CTSP nucleic acids or fragments derivedtherefrom.

Methods for immunization, including formulation of a vaccine compositionand selection of doses, route of administration and the schedule ofadministration (e.g. primary and one or more booster doses), are wellknown in the art. The tests also can be performed in humans, where theend point is to test for the presence of enhanced levels of circulatingCTLs against cells bearing the antigen, to test for levels ofcirculating antibodies against the antigen, to test for the presence ofcells expressing the antigen and so forth.

As part of the immunization compositions, one or more CTSP polypeptidesor immunogenic fragments thereof are administered with one or moreadjuvants to induce an immune response or to increase an immuneresponse. An adjuvant is a substance incorporated into or administeredwith antigen which potentiates the immune response. Adjuvants mayenhance the immunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., 1997, Mol. Cells 7:178-186); incomplete Freund'sadjuvant; complete Freund's adjuvant; montanide; alum; CpGoligonucleotides (see e.g., Kreig et al., 1995, Nature 374:546-9); andvarious water-in-oil emulsions prepared from biodegradable oils such assqualene and/or tocopherol. Preferably, the antigens are administeredmixed with a combination of DQS21/MPL. The ratio of DQS21 to MPLtypically will be about 1:10 to 10:1, preferably about 1:5 to 5:1 andmore preferably about 1:1. Typically for human administration, DQS21 andMPL will be present in a vaccine formulation in the range of about 1 μgto about 100 μg. Other adjuvants are known in the art and can be used inthe invention (see, e.g., Goding, Monoclonal Antibodies: Principles andPractice, 2nd Ed., 1986). Methods for the preparation of mixtures oremulsions of polypeptide and adjuvant are well known to those of skillin the art of vaccination.

Other agents which stimulate the immune response of the subject can alsobe administered to the subject. For example, other cytokines are alsouseful in vaccination protocols as a result of their lymphocyteregulatory properties. Many other cytokines useful for such purposeswill be known to one of ordinary skill in the art, includinginterleukin-12 (IL-12) which has been shown to enhance the protectiveeffects of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSFand IL-18. Thus cytokines can be administered in conjunction withantigens and adjuvants to increase the immune response to the antigens.

There are a number of immune response potentiating compounds that can beused in vaccination protocols. These include costimulatory moleculesprovided in either protein or nucleic acid form. Such costimulatorymolecules include the B7-1 and B7-2 (CD80 and CD86 respectively)molecules which are expressed on dendritic cells (DC) and interact withthe CD28 molecule expressed on the T cell. This interaction providescostimulation (signal 2) to an antigen/MHC/TCR stimulated (signal 1) Tcell, increasing T cell proliferation and effector function. B7 alsointeracts with CTLA4 (CD152) on T cells and studies involving CTLA4 andB7 ligands indicate that the B7-CTLA4 interaction can enhance antitumorimmunity and CTL proliferation (Zheng P. et al., 1998, Proc. Natl. Acad.Sci. U.S.A. 95 (11):6284-6289).

B7 typically is not expressed on tumor cells so they are not efficientantigen presenting cells (APCs) for T cells. Induction of B7 expressionwould enable the tumor cells to stimulate more efficiently CTLproliferation and effector function. A combination of B7/IL-6/IL-12costimulation has been shown to induce IFN-gamma and a Th1 cytokineprofile in the T cell population leading to further enhanced T cellactivity (Gajewski et al., 1995, J. Immunol, 154:5637-5648). Tumor celltransfection with B7 has been discussed in relation to in vitro CTLexpansion for adoptive transfer immunotherapy by Wang et al., (J.Immunol., 19:1-8, 1986). Other delivery mechanisms for the B7 moleculewould include nucleic acid (naked DNA) immunization (Kim J., et al.,1997, Nat. Biotechnol., 15(7):641-646) and recombinant viruses such asadeno and pox (Wendtner et al., 1997, Gene Ther., 4(7):726-735). Thesesystems are all amenable to the construction and use of expressioncassettes for the coexpression of B7 with other molecules of choice suchas the antigens or fragment(s) of antigens discussed herein (includingpolytopes) or cytokines. These delivery systems can be used forinduction of the appropriate molecules in vitro and for in vivovaccination situations. The use of anti-CD28 antibodies to directlystimulate T cells in vitro and in vivo could also be considered.Similarly, the inducible co-stimulatory molecule ICOS which induces Tcell responses to foreign antigen could be modulated, for example, byuse of anti-ICOS antibodies (Hutloff et al., 1999, Nature 397:263-266).

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCsand some tumor cells and interacts with CD2 expressed on T cells. Thisinteraction induces T cell IL-2 and IFN-gamma production and can thuscomplement but not substitute, the B7/CD28 costimulatory interaction(Parra et al., 1997, J. Immunol. 158:637-642; Fenton et al., 1998, J.Immunother., 21(2):95-108).

Lymphocyte function associated antigen-1 (LFA-1) is expressed onleukocytes and interacts with ICAM-1 expressed on APCs and some tumorcells. This interaction induces T cell IL-2 and IFN-gamma production andcan thus complement but not substitute, the B7/CD28 costimulatoryinteraction (Fenton et al., 1998, J. Immunother., 21(2):95-108). LFA-1is thus a further example of a costimulatory molecule that could beprovided in a vaccination protocol in the various ways discussed abovefor B7.

Complete CTL activation and effector function requires Th cell helpthrough the interaction between the Th cell CD40L (CD40 ligand) moleculeand the CD40 molecule expressed by DCs (Ridge et al., 1998, Nature393:474; Bennett et al., 1998, Nature 393:478; Schoenberger et al.,1998, Nature 393:480). This mechanism of this costimulatory signal islikely to involve upregulation of B7 and associated IL-6/IL-12production by the DC (APC). The CD40-CD40L interaction thus complementsthe signal 1 (antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.

The use of anti-CD40 antibodies to stimulate DC cells directly, would beexpected to enhance a response to tumor antigens which are normallyencountered outside of an inflammatory context or are presented bynon-professional APCs (tumor cells). In these situations Th help and B7costimulation signals are not provided.

The invention contemplates delivery of nucleic acids, polypeptides orfragments thereof for vaccination. Delivery of polypeptides andfragments thereof can be accomplished according to standard vaccinationprotocols which are well known in the art. In another embodiment, thedelivery of nucleic acid is accomplished by ex vivo methods, i.e. byremoving a cell from a subject, genetically engineering the cell toexpress or include a CTSP polypeptide, and reintroducing the engineeredcell into the subject. One example of such a procedure is outlined inU.S. Pat. No. 5,399,346 and in exhibits submitted in the file history ofthat patent, all of which are publicly available documents. In general,it involves introduction in vitro of a functional copy of a gene into acell(s) of a subject, and returning the genetically engineered cell(s)to the subject. The functional copy of the gene is under operablecontrol of regulatory elements which permit expression of the gene inthe genetically engineered cell(s). Numerous transfection andtransduction techniques as well as appropriate expression vectors arewell known to those of ordinary skill in the art, some of which aredescribed in PCT application WO95/00654. In vivo nucleic acid deliveryusing vectors such as viruses and targeted liposomes also iscontemplated according to the invention.

A virus vector for delivering a nucleic acid encoding a CTSP polypeptideis selected from the group consisting of adenoviruses, adeno-associatedviruses, poxviruses including vaccinia viruses and attenuatedpoxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus,retroviruses, Sindbis virus, and Ty virus-like particle. Examples ofviruses and virus-like particles which have been used to deliverexogenous nucleic acids include: replication-defective adenoviruses(e.g., Xiang et al., 1996, Virology 219:220-227; Eloit et al., 1997, J.Virol. 7:5375-5381; Chengalvala et al., 1997, Vaccine 15:335-339), amodified retrovirus (Townsend et al., 1997, J. Virol. 71:3365-3374), anonreplicating retrovirus (Irwin et al., 1994, J. Virol. 68:5036-5044),a replication defective Semliki Forest virus (Zhao et al., 1995, Proc.Natl. Acad. Sci. U.S.A. 92:3009-3013), canarypox virus and highlyattenuated vaccinia virus derivative (Paoletti, 1996, Proc. Natl. Acad.Sci. U.S.A. 93:11349-11353), non-replicative vaccinia virus (Moss, 1996,Proc. Natl. Acad. Sci. U.S.A. 93:11341-11348), replicative vacciniavirus (Moss, 1994, Dev. Biol. Stand. 82:55-63), Venzuelan equineencephalitis virus (Davis et al., 1996, J. Virol. 70:3781-3787), Sindbisvirus (Pugachev et al., 1995, Virology 212:587-594), and Ty virus-likeparticle (Allsopp et al., 1996, Eur. J. Immunol. 26:1951-1959). Apreferred virus vector is an adenovirus.

Preferably the foregoing nucleic acid delivery vectors: (1) containexogenous genetic material that can be transcribed and translated in amammalian cell and that can induce an immune response in a host, and (2)contain on a surface a ligand that selectively binds to a receptor onthe surface of a target cell, such as a mammalian cell, and therebygains entry to the target cell.

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection or infection with the foregoingviruses including the nucleic acid of interest, liposome mediatedtransfection, and the like. For certain uses, it is preferred to targetthe nucleic acid to particular cells. In such instances, a vehicle usedfor delivering a nucleic acid of the invention into a cell (e.g., aretrovirus, or other virus; a liposome) can have a targeting moleculeattached thereto. For example, a molecule such as an antibody specificfor a surface membrane protein on the target cell or a ligand for areceptor on the target cell can be bound to or incorporated within thenucleic acid delivery vehicle. Preferred antibodies include antibodieswhich selectively bind a CTSP polypeptide, alone or as a complex with aMHC molecule. Especially preferred are monoclonal antibodies. Whereliposomes are employed to deliver the nucleic acids of the invention,proteins which bind to a surface membrane protein associated withendocytosis may be incorporated into the liposome formulation fortargeting and/or to facilitate uptake. Such proteins include capsidproteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfully to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

The invention further embraces methods for assessing prognosis of asubject diagnosed with cancer, such as cancers that include abnormalCTSP expression. As used herein, “assessing prognosis” means determiningthe likelihood of survival of a subject having or diagnosed with adisorder based on one or more biological and/or pathological parameters.Such prognostic application of the invention is particularly useful forassessing the prognosis for a subject previously diagnosed with acancer, such as a carcinoma, preferably prostate cancer. In case ofprostate cancer, for example, more than one in three patients that haveundergone radical prostatectomy (RRP) show biochemical recurrence withinten years. Therefore, novel biomarkers that can be used to assessprognosis, for example by predicting the risk of biochemical recurrencein cancer patients are of clinical interest.

In general, “biochemical recurrence” is defined as an increase of a PSAlevel in a biological sample to >0.2 ng/mL (Roehl et al., 2004, J Urol.172(3):910-4; Hull et al., 2002, J Urol. 167(2 Pt 1):528-34; Amling etal., 2000 J Urol. 2000, 164(1):101-5; Grossfeld et al., 2003, J Urol.169(1):157-63).

As shown in more detail below, the invention provides methods forassessing the prognosis of a subject with prostate cancer. The methodinvolves obtaining a biological sample, e.g., plasma sample and a biopsysample, from a subject and determining the presence of anti-CTSP-1antibodies in the sample. The CTSP-1 polypeptides described herein, andfragments thereof that bind antibodies, are useful in theimmunodetection of anti-CTSP-1 antibodies in the biological sample. Theabsence of anti-CTSP-1 antibodies in a biological sample is indicativeof poor prognosis. In contrast, the presence of anti-CTSP-1 antibodiesin a biological sample is indicative of good prognosis.

As used herein, “poor prognosis” means that there is a shortened periodbetween the time a patient receives a treatment (e.g., RRP) and the timethe patient presents biochemical recurrence of the disease. As usedherein, “good prognosis” means that there is better outlook of survival,i.e., extended survival, (for example, after five years) among patients,as compared to control patients.

As will be clear to those skilled in the art, in some cases, theprognostic methods provided in the invention may be used in combinationwith other biomarkers of the disease. Accordingly, the methods describedherein may be used, for example, in conjunction with other prostatecancer markers, such as prostate specific antigen (PSA), prostatespecific membrane antigen (PSMA), prostatic acid phosphatase (PAP) orNY-ESO-1 in assessing prognosis. For example, the prognostic methods ofthe invention may further comprise determining the presence, absence orlevel of expression of NY-ESO-1 mRNA and/or NY-ESO-1 polypeptide in abiological sample using detection techniques well known in the art, suchas PCR-based methods and hybridization-based methods for mRNA detectionand immunological methods for detection of polypeptide. In such methods,the presence of NY-ESO-1 mRNA and/or NY-ESO-1 polypeptide in thebiological sample is indicative of poor prognosis. Similarly, in somecases, the methods may comprise determining the presence or absenceanti-NY-ESO-1 antibodies in a biological sample using immunologicaldetection techniques well known in the art. In such methods, thepresence of anti-NY-ESO-1 antibodies in the biological sample isindicative of poor prognosis.

According to a further aspect of the invention, compositions containingthe nucleic acid molecules, proteins, and binding polypeptides of theinvention are provided. The compositions contain any of the foregoingtherapeutic agents in a carrier, optionally a pharmaceuticallyacceptable carrier. Thus, in a related aspect, the invention provides amethod for forming a medicament that involves placing a therapeuticallyeffective amount of the therapeutic agent in the pharmaceuticallyacceptable carrier to form one or more doses. The effectiveness oftreatment or prevention methods of the invention can be determined usingstandard diagnostic methods described herein.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The term “physiologicallyacceptable” refers to a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism. Thecharacteristics of the carrier will depend on the route ofadministration. Physiologically and pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials which are well known in the art. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intratumoral, intraperitoneal, intramuscular, intracavity, subcutaneous,or transdermal. When antibodies are used therapeutically, a preferredroute of administration is by pulmonary aerosol. Techniques forpreparing aerosol delivery systems containing antibodies are well knownto those of skill in the art. Generally, such systems should utilizecomponents which will not significantly impair the biological propertiesof the antibodies, such as the paratope binding capacity (see, forexample, Sciarra and Cutie, “Aerosols,” in Remington's PharmaceuticalSciences, 18th edition, 1990, pp 1694-1712). Those of skill in the artcan readily determine the various parameters and conditions forproducing antibody aerosols without undue experimentation. When usingantisense preparations of the invention, slow intravenous administrationis preferred.

The compositions of the invention are administered in effective amounts.An “effective amount” is that amount of a CTSP polypeptide compositionthat alone, or together with further doses, produces the desiredresponse, e.g. increases an immune response to the CTSP polypeptide. Inthe case of treating a particular disease or condition characterized byexpression of one or more CTSP polypeptides, such as cancer, the desiredresponse is inhibiting the progression of the disease. This may involveonly slowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. This can be monitored by routine methods or can bemonitored according to diagnostic methods of the invention discussedherein. The desired response to treatment of the disease or conditionalso can be delaying the onset or even preventing the onset of thedisease or condition.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the individual components or combinations thereofbe used, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of CTSP polypeptide ornucleic acid encoding CTSP polypeptide for producing the desiredresponse in a unit of weight or volume suitable for administration to apatient. The response can, for example, be measured by determining theimmune response following administration of the CTSP polypeptidecomposition via a reporter system by measuring downstream effects suchas gene expression, or by measuring the physiological effects of theCTSP polypeptide composition, such as regression of a tumor or decreaseof disease symptoms. Other assays will be known to one of ordinary skillin the art and can be employed for measuring the level of the response.

The doses of CTSP polypeptide compositions (e.g., polypeptide, peptide,antibody, cell or nucleic acid) administered to a subject can be chosenin accordance with different parameters, in particular in accordancewith the mode of administration used and the state of the subject. Otherfactors include the desired period of treatment. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits.

In general, for treatments for eliciting or increasing an immuneresponse, doses of CTSP polypeptide are formulated and administered indoses between 1 ng and 1 mg, and preferably between 10 ng and 100 mg,according to any standard procedure in the art. Where nucleic acidsencoding CTSP polypeptides or variants thereof are employed, doses ofbetween 1 ng and 0.1 mg generally will be formulated and administeredaccording to standard procedures. Other protocols for the administrationof CTSP polypeptide compositions will be known to one of ordinary skillin the art, in which the dose amount, schedule of injections, sites ofinjections, mode of administration (e.g., intra-tumoral) and the likevary from the foregoing. Administration of CTSP polypeptide compositionsto mammals other than humans, e.g. for testing purposes or veterinarytherapeutic purposes, is carried out under substantially the sameconditions as described above.

Where CTSP polypeptides are used for vaccination, modes ofadministration which effectively deliver the CTSP polypeptide andadjuvant, such that an immune response to the polypeptide is increased,can be used. For administration of a CTSP polypeptide in adjuvant,preferred methods include intradermal, intravenous, intramuscular andsubcutaneous administration. Although these are preferred embodiments,the invention is not limited by the particular modes of administrationdisclosed herein. Standard references in the art (e.g., Remington'sPharmaceutical Sciences, 18th edition, 1990) provide modes ofadministration and formulations for delivery of immunogens with adjuvantor in a non-adjuvant carrier.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, and lactated Ringer's or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases, and the like.

The pharmaceutical agents of the invention may be administered alone, incombination with each other, and/or in combination with otheranti-cancer drug therapies and/or treatments. These therapies and/ortreatments may include, but are not limited to: surgical intervention,chemotherapy, radiotherapy, and adjuvant systemic therapies.

The invention also provides a pharmaceutical kit comprising one or morecontainers comprising one or more of the pharmaceutical compounds oragents of the invention. Additional materials may be included in any orall kits of the invention, and such materials may include, but are notlimited to buffers, water, enzymes, tubes, control molecules, etc. Thekit may also include instructions for the use of the one or morepharmaceutical compounds or agents of the invention for the treatment ofcancer.

The invention includes kits for assaying the presence of CTSPpolypeptides and/or antibodies that specifically bind to CTSPpolypeptides. An example of such a kit may include the above-mentionedpolypeptides bound to a substrate, for example a dipstick, which isdipped into a blood or body fluid sample of a subject. The surface ofthe substrate may then be processed using procedures well known to thoseof skill in the art, to assess whether specific binding occurred betweenthe polypeptides and agents (e.g., antibodies) in the subject's sample.For example, procedures may include, but are not limited to, contactwith a secondary antibody, or other method that indicates the presenceof specific binding.

Another example of a kit may include an antibody or antigen-bindingfragment thereof, that binds specifically to a CTSP polypeptide. Theantibody, or antigen-binding fragment thereof, may be applied to atissue or cell sample from a patient with cancer and the sample thenprocessed to assess whether specific binding occurs between the antibodyand an antigen or other component of the sample. In addition, theantibody, or antigen-binding fragment thereof, may be applied to a bodyfluid sample, such as serum, from a subject, either suspected of havingcancer, diagnosed with cancer, or believed to be free of cancer. As willbe understood by one of skill in the art, such binding assays may alsobe performed with a sample or object contacted with an antibody and/orCTSP polypeptide that is in solution, for example in a 96-well plate, orapplied directly to a solid support (i.e., an object's surface).

Another example of a kit of the invention is a kit that providescomponents necessary to determine the level of expression of one or moreCTSP nucleic acid molecules of the invention. Such components mayinclude primers useful for amplification of one or more CTSP nucleicacid molecules and/or other chemicals for PCR amplification.

Another example of a kit of the invention is a kit that providescomponents necessary to determine the level of expression of one or moreCTSP nucleic acid molecules of the invention using a method ofhybridization.

The foregoing kits can include instructions or other printed material onhow to use the various components of the kits for diagnostic purposes.

The invention further includes nucleic acid or protein microarrays(including antibody arrays) for the analysis of expression of CTSPpolypeptides or nucleic acids encoding such antigens. In this aspect ofthe invention, standard techniques of microarray technology are utilizedto assess expression of the CTSP polypeptides and/or identify biologicalconstituents that bind such antigens. The constituents of biologicalsamples include antibodies, lymphocytes (particularly T lymphocytes),and the like. Microarray substrates include but are not limited toglass, silica, aluminosilicates, borosilicates, metal oxides such asalumina and nickel oxide, various clays, nitrocellulose, or nylon. Themicroarray substrates may be coated with a compound to enhance synthesisof a probe (peptide or nucleic acid) on the substrate. Coupling agentsor groups on the substrate can be used to covalently link the firstnucleotide or amino acid to the substrate. A variety of coupling agentsor groups are known to those of skill in the art. Peptide or nucleicacid probes thus can be synthesized directly on the substrate in apredetermined grid. Alternatively, peptide or nucleic acid probes can bespotted on the substrate, and in such cases the substrate may be coatedwith a compound to enhance binding of the probe to the substrate. Inthese embodiments, presynthesized probes are applied to the substrate ina precise, predetermined volume and grid pattern, preferably utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate. Nucleic acid probes preferably are linked using UVirradiation or heat.

Protein microarray technology, which is also known by other namesincluding protein chip technology and solid-phase protein arraytechnology, is well known to those of ordinary skill in the art and isbased on, but not limited to, obtaining an array of identified peptidesor proteins on a fixed substrate, binding target molecules or biologicalconstituents to the peptides, and evaluating such binding. See, e.g., G.MacBeath and S. L. Schreiber, “Printing Proteins as Microarrays forHigh-Throughput Function Determination,” Science 289(5485):1760-1763,2000.

Targets are peptides or proteins and may be natural or synthetic. Thetissue may be obtained from a subject or may be grown in culture (e.g.,from a cell line).

In some embodiments of the invention, one or more control peptide orprotein molecules are attached to the substrate. Preferably, controlpeptide or protein molecules allow determination of factors such aspeptide or protein quality and binding characteristics, reagent qualityand effectiveness, hybridization success, and analysis thresholds andsuccess.

Nucleic acid arrays, particularly arrays that bind CTSP nucleic acidsequences, also can be used for diagnostic applications, such as foridentifying subjects that have a condition characterized by aberrantCTSP molecule expression, e.g., cancer. Nucleic acid microarraytechnology, which is also known by other names including: DNA chiptechnology, gene chip technology, and solid-phase nucleic acid arraytechnology, is well known to those of ordinary skill in the art and isbased on, but not limited to, obtaining an array of identified nucleicacid probes on a fixed substrate, labeling target molecules withreporter molecules (e.g., radioactive, chemiluminescent, or fluorescenttags such as fluorescein, Cye3-dUTP, or Cye5-dUTP), hybridizing targetnucleic acids to the probes, and evaluating target-probe hybridization.A probe with a nucleic acid sequence that perfectly matches the targetsequence will, in general, result in detection of a strongerreporter-molecule signal than will probes with less perfect matches.Many components and techniques utilized in nucleic acid microarraytechnology are presented in The Chipping Forecast, Nature Genetics, Vol.21, January 1999, the entire contents of which is incorporated byreference herein.

According to the invention, probes are selected from the group ofnucleic acids including, but not limited to: DNA, genomic DNA, cDNA, andoligonucleotides; and may be natural or synthetic. Oligonucleotideprobes preferably are 15 to 40-mer oligonucleotides and DNA/cDNA probespreferably are 200 to 5000 bases in length, although other lengths maybe used. Appropriate probe length may be determined by one of ordinaryskill in the art by following art-known procedures. In one embodiment,preferred probes are sets of one or more of the CTSP nucleic acidmolecules as described herein. Probes may be purified to removecontaminants using standard methods known to those of ordinary skill inthe art such as gel filtration or precipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or oligonucleotide to thesubstrate. These agents or groups may include, for example, amino,hydroxy, bromo, and carboxy groups. These reactive groups are preferablyattached to the substrate through a hydrocarbyl radical such as analkylene or phenylene divalent radical, one valence position occupied bythe chain bonding and the remaining attached to the reactive groups.These hydrocarbyl groups may contain up to about ten carbon atoms,preferably up to about six carbon atoms. Alkylene radicals are usuallypreferred containing two to four carbon atoms in the principal chain.These and additional details of the process are disclosed, for example,in U.S. Pat. No. 4,458,066, which is incorporated by reference in itsentirety.

In one embodiment, nucleic acid probes are synthesized directly on thesubstrate in a predetermined grid pattern using methods such aslight-directed chemical synthesis, photochemical deprotection, ordelivery of nucleotide precursors to the substrate and subsequent probeproduction.

Targets for microarrays are nucleic acids selected from the group,including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and maybe natural or synthetic. In all embodiments, nucleic acid targetmolecules from human tissue are preferred. The tissue may be obtainedfrom a subject or may be grown in culture (e.g., from a cell line).

In embodiments of the invention one or more control nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as nucleic acidquality and binding characteristics, reagent quality and effectiveness,hybridization success, and analysis thresholds and success. Controlnucleic acids may include but are not limited to expression products ofgenes such as housekeeping genes or fragments thereof.

EXAMPLES

Materials and Methods

Generation of CTSP-1 Full-Length Sequence

cDNA sequencing: C21ORF99 sequence (SEQ ID NO: 12) was initiallyobtained by sequencing the full insert of cDNA clones from which theESTs aligning to human chromosome 21 (HC21) were derived. cDNA clones(IMAGE 1461135 and 2909444) were obtained from Research Genetics(Invitrogen Corporation, Carlsbad Calif.) and were sequenced directlyusing the vector's primers.

RT-PCR: NYBR-1 and NYBR1.1 sequences were aligned to HC21 genomicsequence using the BLASTN program. Primers for RT-PCR were designedbased on the HC21 genomic sequence in regions that showed similarity toNYBR-1 and NYBR-1.1 sequences and that were not covered by the C21ORF99sequence. Primers were manually designed in order to avoid unspecificamplification of NYBR-1 or NYBR-1.1. RT-PCR reactions were carried outin a 25 μL reaction containing 1 μl of first strand testis cDNA, 1× TaqDNA polymerase buffer (Invitrogen, Carlsbad, Calif.), 0.1 mM dNTP, 2 mMMgCl₂, 1 unit Taq DNA polymerase (Invitrogen) and 6 pmol of thefollowing primers CTSP-F1 5′ CTGAAAGCTTGGTGGAAAG 3′ (SEQ ID NO: 25) andCTSP-R1 5′ GTTCCTTCTTCCAAAACTTC 3′ (SEQ ID NO: 26) or CTSP-F2 5′AAGACTGAATGAGTGGCAG 3′ (SEQ ID NO: 27) and CTSP-R2 5′CTGATTCAAATTACTTCTTACAG 3′(SEQ ID NO: 28). Amplification conditionswere: initial denaturation for 4 min at 94° C. followed by 40 cycles of45 sec at 94° C., 45 sec at 58° C. and 1 min at 72° C. and a finalextension of 10 min at 72° C. PCR products were analyzed on 8%silver-stained polyacrylamide gels and cloned using the TA cloning kit(Invitrogen). Individual colonies were sequenced using Dynamic ETterminator cycle sequencing (Amersham, Piscataway, N.J.) and an ABI3100Prism sequencer. Sequences were aligned to HC21 sequence using BLASTN toconfirm their specificity.

Rapid Amplification of cDNA Ends: 3′ RACE was performed on normal testispoly(A)+ RNA using the Marathon cDNA Amplification Kit (Clontech,Mountain View, Calif.). Double-strand cDNA synthesis and adaptorsligation to the synthesized cDNAs were carried out according to themanufacturers' instructions. Amplification reactions were performed in a25 μl reaction using 5 μl of cDNA, 0.2 mM dNTPs, 0.2 μM of CTSP-1specific primer (SP-RACE 5′ CCATGGCTCACACCTGTAATCTCATCAC 3′; SEQ ID NO:29), 0.2 μM of the adaptor primers (ADPT 5′CGACGTGGACTATCCATGAACGCACGCAGTCGGTAC(T)₁₃ 3′; SEQ ID NO: 30) and 1 unitof Advantage Taq DNA polymerase (Clontech). PCR conditions were: 1 min.at 94° C. followed by 5 cycles of 5 sec at 94° C. and 4 min at 70° C., 5cycles of 5 sec at 94° C. and 4 min at 68° C., and 25 cycles of 5 sec at94° C., 30 sec at 65° C. with a final extension step of 4 min at 68° C.Nested-PCR was carried out in the same conditions using 1 μl of thefirst reaction product and the following primers: SP-RACE3N 5′GAAAAAGTTAGAAGTGAAGCAACTTGAG 3′ (SEQ ID NO: 31) and ADPTN 5′TCGAGCGGCCGCCCGGGCAGGTCGACGTGGACTATCCATGAACGCA 3′(SEQ ID NO: 32).

PCR products were cloned using the TA cloning kit (Invitrogen) andsequenced as described above.

Computational Analysis

The CTSP-1 consensus sequence was obtained by assembling the C21ORF99sequence, the two RT-PCR fragments and the 3′ RACE fragments usingPhred/Phrap/Consed (Gordon et al., 1998, Genome Res. 8:195-202).Repetitive elements along the CTSP-1 sequence were identified withRepeatMasker (A. F. A. Smit, R. Hubley & P. Green RepeatMasker athttp://repeatmasker.org). CTSP family members and partial copies wereidentified by aligning CTSP-1 consensus sequence against the humangenome assembly (May 2004) available at the UCSC Genome Browser usingBLAT (Kent, 2002, Genome Res. 12:656-664). CTSP-2, 3 and 4 exon-intronstructure and consensus sequences were predicted by pairwise alignmentsof the CTSP-1 consensus sequence with the human genome sequence usingSim4 (Florea et al., 1998, Genome Res. 8:967-974). Consensus sequencesincluding all predicted exons were translated in all six reading framesand putative proteins were aligned using ClustalW (Higgins et al., 1996,Methods Enzymol. 266: 383-402). Protein motif searches were performedwith PROSITE (Sigrist et al., 2002, Brief Bioinform. 3: 265-274) andPfam databases (Bateman et al., 2004, Nucleic Acids Res. 32: D138-D141).

Expression Analysis

Samples: Human tumor cell lines A172 and T98G (glioblastoma); FaDu(squamous cell carcinoma); SW480 (colorectal adenocarcinoma); Skme1-28and A2058 (malignant melanoma); DU145 and PC3 (prostate carcinoma); HeLaand CasKi (cervix adenocarcinoma); MDA-MB-231, MCF-7 and MDA-MB-436(breast ductal carcinoma); IM9 (B transformed lymphoblasts); HL60 andK562 (lymphocytes); Help G2 (hepatocarcinoma); H1155 and H358 (lungcarcinoma); SCABER (urinary bladder carcinoma); SAOS-2 (osteosarcoma)were obtained from the American Type Culture Collection (ATCC) andmaintained in appropriate medium as recommended by this organization(atcc.org). Tumor samples were collected from patients treated atHospital A.C. Camargo. All samples were collected after explicitinformed consent and with local ethical committee approval. Total RNAderived from 21 normal human tissues (testis, lung, prostate, smallintestine, breast, brain, heart, uterus, bone marrow, placenta, colon,fetal brain, liver, fetal liver, thymus, salivary gland, spinal cord,kidney, spleen, skeletal muscle, and adrenal gland) was purchased fromClontech.

RNA extraction, cDNA synthesis and RT-PCR: Total RNA was extracted bythe conventional CsCl-guanidine thiocyanate gradient method (MacDonaldet al., 1987, Methods Enzymol. 152: 219-227). RNA samples were checkedfor integrity by agarose gel electrophoresis and 2 μg were used for cDNAsynthesis. Reverse transcription was performed with DNA-free RNA usingSUPERSCRIPT II Reverse Transcriptase (Invitrogen) and oligo dT. Primersused for expression analysis of CTSP-1, CTSP-2, CTSP3 and CTSP-4 were:

(SEQ ID NO: 33) CT1F 5′ GCTGTCCATTATGCTGTTAAC 3′, (SEQ ID NO: 34) CT1R5′ TTTTGAGAATTTTTAGATATC 3′, (SEQ ID NO: 35) CT2F 5′CCTGTGGTGCAGACATCG 3′, (SEQ ID NO: 36) CT2R 5′ATTCCAAAAGTTGTTGATGAAC 3′, (SEQ ID NO: 37) CT3F 5′CTGTCCATTATGCTGTTTATG 3′, (SEQ ID NO: 38) CT3R 5′AATTTTTAGATATCTTTTGTTTG 3′, (SEQ ID NO: 39) CT4F 5′TGCTGATCCAAATATTGTAGG 3′ and (SEQ ID NO: 40) CT4R 5′CATTTAAACTTATCAACTGCAA 3′.

PCR fragments were analyzed either on agarose gels followed by Southernblot analysis or on 8% silver-stained polyacrylamide gels. PCR fragmentswere cloned and sequenced as mentioned above to confirm theirspecificity.

5-Aza 2′Deoxycytidine Treatment

MCF-7 breast tumor cell line was treated with 30 μM of 5-Aza2′deoxycytidine for 48 hours. After treatment the cells were harvestedand used for RNA extraction and cDNA synthesis as described above.RT-PCR amplifications were carried out using the same CTSP-1 primersused for gene expression analysis. Amplification products were analyzedon 8% silver-stained polyacrylamide gels.

CTSP-1 Protein Detection

CTSP-1 recombinant protein: CTSP-1 longest ORF was amplified from normaltestis cDNA using the following primers:

CTSP1RecF 5′ CGGGATCCATGAAGA AGACGACAATG 3′ (SEQ ID NO: 41) andCTSP1RecR 5′ CGGAATTCCTATTCGTCAGGTGTTCT 3′ (SEQ ID NO: 42). The PCRproduct was digested with BamH I and EcoR I and cloned into theexpression vector pET28a (Stratagene). After sequencing, the recombinantplasmid pET28a/CTSP-1 was transformed into E. coli BL-21 A494. Afterinduction with 1 mM IPTG at 37° C. for 4 hours the CTSP-1 recombinantprotein fused with a His-tag was purified by Ni²⁺ affinitychromatography using the NiNTA agarose resin (Invitrogen). The purifiedprotein was analyzed by Western blot using an anti-His-tag monoclonalantibody (Amersham Biosciences) to confirm the purification specificity.

CTSP-1 polyclonal antibody: C57 mice (3-months old females) wereimmunized subcutaneously with 25 μg of CTSP-1 recombinant protein usingFreund's Complete Adjuvant (Sigma, St. Louis, Mo.) and Mg(OH)₂ (EMS).Three weeks latter, the mice were boosted with a subcutaneous injectionof 25 μg of the recombinant protein using Freund's Incomplete Adjuvantand a final boost was repeated 3 weeks later. Two weeks after the finalboost, the sera were collected and analyzed by ELISA to determineantibody titer. A total of 250 ng/well of CTSP-1 recombinant in coatingbuffer (0.015M Sodium Carbonate and 0.031M Sodium Bicarbonate, pH 9.6)were adsorbed to ELISA plates (DYNEX Immulon® 2HB) overnight at 4° C.Plates were washed with PBS and blocked with 5% BSA in PBS-Tween (10 mMphosphate buffer, 140 mM NaCl, 0.05% Tween® 20, pH 7.4). The sera werediluted with PBS-Tween plus 0.1% BSA and incubated on the plates for 1hour at room temperature. After washing with PBS-Tween, the boundantibodies were detected with peroxidase-conjugated anti-mouse IgGantibody (1:10000, Amersham Biosciences) using SIGMA FAST™ OPD tabletsas substrate (Sigma).

Western blot: Normal testis tissue was homogenized in sample buffer (240mM Tris pH 6.8, 0.8% SDS, 200 mM β-mercaptoethanol, 40% glycerol and0.2% bromophenol blue). A total of 100 μg of the lysate was loaded on12% SDS/PAGE gels and after fractionation transferred to Hybond-P PDVFmembranes (Amersham Biosciences). After blocking with PBS solutioncontaining 5% low-fat milk, the membranes were incubated with mice seraat a 1:10000 dilution for 1 hour at room temperature. Sera antibodiesbinding to CTSP-1 protein were detected by incubation with rabbitanti-mouse IgG HRP-conjugate (Amersham Biosciences) and visualized withECL™ Western Blotting Detection Reagents (Amersham Biosciences).

Immunohistochemistry

Normal testis and prostate specimens were fixed with buffered formalinand embedded in paraffin. Sections of 3 μm were placed on glass slides,heated at 60° C. for 20 minutes and then placed three times in asolution of xylene for 5 min, four times in 100% methanol for 30 sec andthen washed in water rinses for 5 min. Slides were placed three times in3% hydrogen peroxide for 5 min and washed in water rinses for 5 minprior to incubation for 24 hours in a humidified chamber with theprimary antibody diluted 1:100. Slides were washed in PBS, incubatedwith biotinylated goat antirabbit IgG for 20 min and then withstreptavidin-biotin peroxidase LSAB kit (Dako®, Carpinteria, USA) in ahumidified chamber. The immunostaining was performed by incubatingslides in diaminobenzidine (Dako) solution containing 1 μl of chromogenfor 50 μl of buffer substrate during 5 min. After chromogen development,slides were washed, dehydrate with alcohol and xylene, and mounted withcoverslips using a permanent mounting medium.

Antibody Response in Cancer Patients

Plasmas were obtained from patients with various tumor types (breast,colon, esophagus, lung, melanoma, prostate, stomach, thyroid and uterus)treated at the Hospital A.C. Camargo. All samples were collected afterexplicit informed consent and with local ethical committee approval. Inaddition, plasma samples from 50 healthy individuals were collected fromblood donors at the Hospital A.C. Camargo Blood Center. Antibodiesagainst CTSP-1 recombinant protein were detected by Western blot. Fivehundred nanograms of purified CTSP-1 recombinant protein were fractionedon 12% SDS-PAGE gel electrophoresis and transferred to Hybond-P PVDFmembranes. After blocking with PBS solution containing 5% low-fat milk,the membranes were incubated for 1 hour at room temperature with plasmafrom healthy individuals or cancer patients at a 1:25 dilution. Plasmaantibodies binding to CTSP-1 protein were detected by incubation withgoat anti-human IgG HRP-conjugate (Amersham Biosciences) and visualizedwith ECL™ Western Blotting Detection Reagents (Amersham Biosciences).

Example 1 Identification and Expression of CTSP-1

CTSP-1 Gene Structure and Alternative Polyadenylation Isoforms

Using the information generated by alignments between expressed sequencetags (ESTs) and the genomic sequence of the human chromosome 21 (HC21)(Reymond et al, 2002, Genomics 79: 824-832), a gene was identified andhas been named C21ORF99 (AF427490; SEQ ID NO: 12), which waspredominantly expressed in normal testis and showed high similarity tothe amino-terminal region of the breast differentiation tumor antigenNYBR-1 and to its paralog NYBR-1.1 located at chromosomes 10 and 18,respectively (SEQ ID NO: 24). NYBR-1 cDNA was isolated by serologicalscreening of a breast cancer library and expression analysis reveled arestricted mRNA expression in normal breast and testis tissues (Jager etal., 2001, Cancer Res. 61:2055-2061). NYBR-1 expression was also foundin 21 of 25 breast tumors but in only 2 of 82 nonmammary tumors.NYBR-1.1 shares 54% amino acid identity with NYBR-1 and shows also atissue restricted mRNA expression. However, unlike NYBR-1, NYBR-1.1 isexpressed in normal brain in addition to breast and testis (Jager etal., 2001, Cancer Res. 61: 2055-2061).

C21ORF99 sequence was initially obtained by sequencing the full insertof cDNA clones from which the ESTs aligning to HC21 were derived(Reymond et al, 2002, Genomics 79: 824-832). However, based on themapping of NYBR-1 and NYBR-1.1 to HC21 genomic sequence, it wassuspected that the initial sequence obtained for C21ORF99 (derived fromcDNA clones containing both polyA signal and tail) corresponded to ashorter alternative polyadenylation isoform (FIG. 1).

A combination of RT-PCR and 3′ RACE experiments was used to extend theinitial C21ORF99 sequence and to verify the existence of additionalpolyadenylation isoforms. First, two pairs of primers were designedwhich are specific for HC21 sequence in regions that showed similarityto NYBR-1 and NYBR-1.1. RT-PCR products obtained with these primers werecloned and sequenced and the original C21ORF99 sequence was extended asillustrated in FIG. 1.

Since the 3′ end of NYBR-1 (from nt 3,199 to 4,458) and NYBR-1.1 (fromnt 3,220 to 3,673) did not show significant similarity to the HC21sequence, 3′ RACE experiments were performed in order to generate afull-length sequence. Two distinct 3′ RACE fragments corresponding totwo additional alternative polyadenylation isoforms were amplified andsequenced (FIG. 1). All 3′ ESTs available in public databases andcorresponding to CTSP-1 are derived from the shortest isoform suggestingthat it is the most abundantly expressed isoform (data not shown).

A consensus mRNA sequence of 3,915 bp (SEQ ID NO:1), organized in 15exons (SEQ ID NOs:43-57), was obtained by assembling the C21ORF99sequence, the RT-PCR products and the longest 3′ RACE fragment (FIG. 1)and was named CTSP-1. Careful analysis of this consensus sequencerevealed the presence of two transcribed repetitive elements locatedwithin exons 9 (LTR repeat) and 12 (Alu repeat), which could not befound in the NYBR-1 and NYBR-1.1 sequences (FIG. 1).

Interestingly, since the polyadenylation sites associated with theshorter polyadenylation isoform are contained within the LTR repeat, itis tempting to speculate that the presence of this repetitive elementwithin exon 9 is associated with the occurrence of alternativepolyadenylation. It is well known that the insertion of retroelements inthe human genome can affect gene expression by sequence disruption;however it has recently been shown that these sequences can alsomodulate gene expression by introducing intragenic polyadenylationsignals (Roy-Engel et al., 2005, Cytogenet. Genome Res. 110: 365-371).

CTSP-1 Gene Family

The CTSP-1 sequence was aligned to the human genome sequence in order toidentify additional family members. Three complete copies of highlysimilar genes were found on chromosomes 2 (CTSP-2, 98.0% identity), 14(CTSP-3, 95.4% identity) and 18 (CTSP-4, 98.3% identity). Theexon-intron structure seems to be highly conserved among these 4 membersincluding the presence of both LTR and Alu repeats what suggests thatthey are products of recent gene-duplication events (FIG. 2). However,since the exon-intron structure for these loci were predicted based onthe alignment of CTSP-1 to the genomic sequence, confirmation of thisstructure will depend on the generation of expressed sequences coveringthe full extension of all these genes. Recent duplication events havealso been described for other CT antigens such as NY-ESO-1 and SSX,resulting in two or more identical copies with identical codingsequences (Aradhya et al., 2001, Hum. Mol. Genet. 10: 2557-2567; Gure etal., 2002, Int. J. Cancer 101: 448-453). ESTs and/or cDNA clonescorresponding to all these family members can be found in publicdatabases suggesting that all of them are transcribed. As for CTSP-1,all ESTs and cDNA clones corresponding to these family members representthe first 9 exons and are probably derived from the shorterpolyadenylation isoform.

In addition to these complete copies, two partial copies were identifiedin tandem but in opposite directions on a region spanning ˜318 kb onchromosome 9. These partial copies also contain the LTR and Alu repeatsalthough, their exon-intron structure is slightly different and exons 6,8, 14 and 15 are missing when compared to the complete copies (FIG. 2).Additionally, partial copies covering less than 58% of the CTSP-1 mRNAlength and with a lower similarity (<95%) were found on chromosomes 22,15 and 10 (FIG. 2) and they probably result from failed duplicationevents, as has also been observed for other CT-gene families, such asSSX. An extended analysis of the evolution and conservation of this genefamily among primates will be presented elsewhere.

Expression Profile and Alternative Splicing Isoforms

Since one of the criteria for identifying CT antigen genes is theirspecific expression in tumors, but not in normal tissues except intestis, the mRNA expression pattern of CTSP-1 was determined by RT-PCR.Primers used in the expression analysis were manually designed to assurespecific amplification and RT-PCR products were sequenced to confirmtheir specificity. A panel of cDNA samples was analyzed, including 21samples derived from normal tissues, 21 derived from tumor cell linesand 169 derived from tumors of 10 different histological types.

CTSP-1 mRNA expression was first examined by RT-PCR in normal testiscDNA. A pair of primers located in exons 3 and 8 was used in the RT-PCRwith 40 cycles of amplification. Three predominant fragments of 239, 376and 402 bp were obtained from testis cDNA (FIG. 3A) and the wholeamplification product was cloned without prior gel purification. Cloneswith different insert sizes were selected for sequencing. Using thisstrategy we were able to identify at least 8 splicing variants in whichexons 4, 5 and 7 were alternatively spliced as illustrated in FIG. 3B.

CTSP-1 mRNA expression was then analyzed in cDNA samples derived from 20additional normal tissues and 21 tumor cell lines. In order to favor thedetection of all splicing variants, RT-PCR products were transferred tonylon membranes and hybridized with a cDNA probe corresponding to exon3, which is present in all splicing isoforms. Among the 21 normaltissues, CTSP-1 expression was restricted to testis and could also bedetected in 47.6% (10/21) of all analyzed tumor cell lines (FIG. 4).Interestingly, although the expression of several alternative splicingisoforms was detected in normal testis cDNA a less complex pattern ofalternative splicing could be visualized in cDNA derived from tumor celllines (FIG. 3A). The shortest isoform, in which exons 4, 5 and 7 areskipped, appears to be the most abundant and frequently expressedisoform. Alternative splicing is an important regulatory mechanism thatincreases the diversity of proteins transcribed from a single gene. Thisprocess seems to be particularly important in testis development andspermatogenesis since a higher proportion of alternative splicingisoforms has been described in testis (Huang et al., 2005, J. Androl.26:189-196).

The expression profile of CTSP-1 was then analyzed in 169 tumor samplesderived from 10 different histological types of tumors. Positiveexpression was detected in 44.4% (75/169) of the samples (Table 1). Thehighest percentages of positive expression were observed in melanomas(59.0%) followed by prostate (58.0%) and lung (57%) tumors. As observedin the tumor cell lines, the shorter isoform was the most abundant andfrequently expressed. This expression pattern is partially in agreementwith the classification proposed by Scanlan et al. (Scanlan et al.,2004, Cancer Immun. 23(4):1. Review). In the proposed classification,melanomas and lung tumors were defined as tissues with higher CTexpression tissues; breast, prostate and esophagus were classified ashaving moderate CT expression and stomach and colon as having low CTexpression. Gliomas, thyroid and uterus tumors were not classified inthis analysis due to the insufficient number of samples analyzed.

The expression pattern of the other 3 family members was also analyzedby RT-PCR. Primers used in the expression analysis were also manuallydesigned to assure specific amplification and RT-PCR products weresequenced to confirm their specificity. Although the expression of theother CTSP-1 family members was also restricted among normal tissues,they were not frequently expressed in tumor cell lines and tumorsamples. This fact has also been observed for other CT-antigen membersof the MAGE and SSX families (Gure et al., 1997, Int. J. Cancer 72:965-971; Lucas et al., 1999, Cancer Res. 59: 4100-4103; Pold et al.,1999, Genomics 59:161-167). A positive expression in normal testis wasdetected for CTSP-2 and 4, the first also being expressed in brain,fetal brain and spinal cord among the 21 normal tissues analyzed.Expression of CTSP-2 and CTSP-4 was not detected in any of the tumorcell lines, although CTSP-2 expression was detected in tumor samples ata lower frequency when compared to CTSP-1 (Table 1). CTSP-3 expressionwas not detected in any of the samples analyzed. Alternative splicingisoforms were also detected for CTSP-2 and CTSP-4.

TABLE 1 Frequency of mRNA expression of CTSP family members in tumorsamples. Tumor CTSP-1 CTSP-2 CTSP-4 Breast 9/25 (36.0%) 1/6 (16.5%) 0/6(0.0%) Colon 7/18 (39.0%) 0/14 (0.0%) 0/14 (0.0%) Esophagus 2/5 (40.0%)2/4 (50.0%) 0/4 (0.0%) Glioblastoma 6/13 (46.0%) not done not done Lung8/14 (57.0%) 2/11 (18.8%) 0/11 (0.0%) Melanoma 10/17 (59.0%) 1/9 (11.0%)0/9 (0.0%) Prostate 14/24 (58.0%) 8/17 (47.0%) 0/17 (0.0%) Stomach 4/9(44.0%) 1/3 (33.3%) 0/3 (0.0%) Thyroid 7/24 (29.0%) 0/6 (0.0%) 0/6(0.0%) Uterus 8/20 (40.0%) 4/14 (28.5%) 0/14 (0.0%) Total 75/169 (44.4%)19/84 (22.6%) 0/84 (0.0%)

An important element in the induction of CT antigen gene expression ispromoter demethylation. It has been shown that demethylation of CT genepromoters with 5-Aza-2′deoxycytidine induces antigen expression in cellsthat do not normally produce them (Weber et al., 1994, Cancer Res. 54:1766-1771; De Smet et al.,1999, Mol. Cell Biol. 19: 7327-7335; Coral etal., 2002, Clin. Cancer Res. 8: 2690-2695). CTSP-1 gene expression wasinduced in the MCF-7 breast tumor cell line after treatment with5-Aza-2′deoxycytidine, suggesting that methylation indeed plays animportant role in CTSP-1 gene expression regulation (FIG. 7).

CTSP-1 Protein

The CTSP-1 sequence was translated to amino acid sequence and thepresence of a stop codon was detected in exon 4, generating a 115 aminoacid-long putative protein (SEQ ID NO:13), which is much shorter thanthe NYBR-1 and NYBR-1.1 proteins. The presence of “premature” stopcodons was also detected in all splicing variants, generating small“truncated” proteins of size ranging from 115 to 202 amino acids (FIG.3B, SEQ ID NOs:14-20). Interestingly, the longest open reading frame waspredicted from the shortest splicing variant (variant h, without exons4, 5 and 7), which is the most frequently expressed isoform in testis,tumor cell lines and tumor samples (FIG. 3).

All other members of the family seem to have “premature” stop codons asevaluated by aligning the CTSP-1 consensus sequence to the genomicsequence of chromosomes 2, 14 and 18. All exons predicted by thealignments were translated to protein. Since exon-intron structure forthese loci were only predicted based on the alignment of CTSP-1 to thegenomic sequence, confirmation of putative protein structures willdepend on the generation of expressed sequences from these genes.

The existence of the CTSP-1 protein was confirmed by Western blotexperiments using a polyclonal antibody generated against the CTSP-1recombinant protein. As shown in FIG. 5A, a single band with theexpected molecular mass (˜22 kDa) was detected in total protein extractsfrom normal testis. No higher molecular mass bands were detected.

CTSP-1 protein was also detected by immunohistochemistry in normaltestis and in a prostate tumor sample. CTSP-1 exhibited an intensestaining in testis and the staining was restricted to germ cells. Noreactivity in other structures of the testis was observed. Among thegerm cells, an intense staining was detected in both the nucleus and thecytoplasm of the cells in later stages of differentiation such asspermatocytes and spermatids (FIG. 5B). It should be noted, however,that spermatogonias were mostly negative. A similar expression patternhas been observed for other non-X CT antigens (CT antigens mappedoutside the chromosome X) (Simpson et al., 2005, Nat Rev Cancer.5(8):615-25. Review). In the prostate tumor sample, staining wasrestricted to the tumor cells and the stroma, as well as the adjacentnormal tissue were not stained by the anti-CTSP-1 antibody (FIG. 5B).Due to the high similarity observed among all family members, apossibility remains that the Western blot and immunohistochemistryresults represent the accumulated expression of all or some of the CTSPmembers.

Structural analysis of NYBR-1 and NYBR-1.1 putative protein sequencesrevealed the presence of a bipartite nuclear signal and of a bZIP site(DNA-binding site followed by leucine zipper motif) suggesting thatNYBR-1 and NYBR-1.1 are transcription factors (Jager et al., 2001,Cancer Res. 61: 2055-2061). NYBR-1 and NYBR-1.1 protein also presentfive tandem ankyrin repeats and tandem repetitive elements of unknownfunction, implying a role in protein-protein interactions. Motifanalysis of the amino acid sequence corresponding to the longest openreading frame of CTSP-1 identified a bipartite nuclear localizationsignal (3-19 amino acids) and four tandem ankyrin repeats (14-47, 48-80,81-103, 114-146 amino acids; these amino acid numbers correspond to thenumbering of the longest protein, SEQ ID NO:20). However, the bZIP siteand the tandem repetitive elements could not be found in the CTSP-1sequence, suggesting that CTSP-1 is a nuclear protein that retained theability to interact with other proteins but had lost the ability tointeract with DNA. A similar protein structure is present in the otherCTSP-1 family members.

Example 2 Humoral Response Against CTSP-1 in Cancer Patients

To evaluate the presence of antibodies against proteins from the CTSP-1gene family in plasma from cancer patients, we established animmunoblotting assay using recombinant his-tagged CTSP-1. Anotherhis-tagged unrelated recombinant protein purified under the sameconditions served as internal negative control.

A total of 141 plasma samples from cancer patients with differenthistological types of tumors were used in the immunoblotting assay(Table 2, FIG. 6). Among the 141 plasmas analyzed, 14 (10.0%) werereactive to the CTSP-1 recombinant protein. Plasma samples from 50healthy donors were used as negative controls. Reactivity against CTSP-1recombinant protein was only detected in one sample from this controlgroup. Antibodies against CT antigens have been detected in patientswith autoimmune disorders such as lupus erythematosus (McCurdy et al.,1998, Mol. Genet. Metab. 63: 3-13) and vitiligo (Rocha et al., 2000,Int. J. Dermatol. 39: 840-843) as well as in men subjected to vasectomy(Lea et al., 1997, Fertil. Steril. 67: 355-361). In addition, apossibility remains that a not yet diagnosed cancer patient within thehealthy control group has been identified.

The highest percentages of antibody response against CTSP-1 wereobserved in patients with prostate (20.8%), thyroid (20.0%) and breast(16.6%) tumors (Table 2). With the exception of NY-ESO-1, humoral immuneresponses to CT antigens are relatively rare, occurring in less than 10%of the cancer patients (Stockert et al., 1998, Exp. Med. 187:1349-1354). Serum antibody to NY-ESO-1 has been detected at a higherfrequency in ovarian cancer, melanoma and breast cancer (Stockert etal., 1998, J. Exp. Med. 187: 1349-1354; Yakirevich et al., 2003, Clin.Cancer Res. 9: 6453-6460; Sugita et al., 2004, Clin. Cancer Res. 9:6453-6460; Jager et al., 1999, Int. J. Cancer 84: 506-510).

TABLE 2 Frequency of anti-CTSP-1 antibodies in plasma samples fromcancer patients. Tumor Antibody Response Breast 3/18 (16.6%) Colon 0/20(0.0%) Esophagus 0/4 (0.0%) Lung 1/13 (8.0%) Melanoma 1/22 (4.5%)Prostate 5/24 (20.8%) Stomach 1/8 (12.5%) Thyroid 2/10 (20.0%) Uterus1/22 (4.5%) Total 14/141 (10.0%)

For 121 out of the 141 patients included in this study, a correlationbetween CTSP-1 mRNA expression and the presence of a humoral immuneresponse could be analyzed since both RNA and plasma samples wereavailable (Table 3). A significant percentage (15.5%) of patientsbearing CTSP-1 expressing tumors developed a humoral immune responseagainst the protein. However, antibodies against CTSP-1 were alsodetected in 6.3% of patients with CTSP-1 negative tumors.

TABLE 3 Correlation between CTSP-1 mRNA expression and CTSP-1 antibodyresponse in cancer patients. CTSP-1 mRNA expression was evaluated byRT-PCR (RT) and the presence of antibodies (Ab) was evaluated by Westernblot. Tumor RT+/Ab+ RT+/Ab− RT−/Ab+ RT−/Ab− Total Breast 2 4 3 6 15Colon 3 4 1 9 17 Esophagus 0 2 0 2 4 Lung 1 7 0 5 13 Melanoma 1 8 0 7 16Prostate 7 4 1 9 21 Stomach 0 4 1 3 8 Thyroid 1 3 1 3 8 Uterus 1 6 0 1219 Total 16 42 7 56 121

A possible explanation for this apparent discrepancy would be thepresence of an immune response against other CTSP family members. Theexpression of CTSP2 and CTSP-4 in these CTSP-1 negative tumors was thenassayed by RT-PCR. Of the 7 CTSP-1 negative tumors analyzed, only oneshowed expression of CTSP-2 and none of them showed expression ofCTSP-4. CTSP-2 and CTSP-4 mRNA expression were also analyzed in 84 ofthe 121 samples from which the corresponding plasma was available. Thesmall percentage of positive expression of CTSP-2 (22.6%) and CTSP4 (0%)in these samples suggests that the immune response observed in thesepatients is likely to be directed to CTSP-1. Alternative explanationsfor the discrepancy between CTSP-1 expression and antibody responsewould be the presence of tumor heterogeneity not represented in thesmall tissue fragment analyzed by RT-PCR; the presence of undetectedmetastases expressing CTSP-1 or the clearance of CTSP-1 positive tumorcells by the immune system.

Example 3 Antibodies Against the Cancer-Testis Antigen CTSP-1 as anIndependent Prognostic Factor for Prostate Cancer

Introduction

Prostate cancer is a significant health problem that is common in menabove the age of 50. It is the most commonly diagnosed malignancy, andthe second leading cause of cancer-related death in men in the UnitedStates (Jemal et al., 2006, CA Cancer J Clin. 56(2):106-30). Radicalprostatectomy (RRP) is the most common and effective treatment forclinically localized prostate cancer. However, approximately 35% ofprostate cancer patients will have biochemical recurrence of the diseasewithin 10 years after surgery. Subsequently, these patients will developclinical relapse of the disease typically featuring bone metastases.

The ability to predict biochemical recurrence and cancer progressionafter RRP using clinical and pathological variables has been extensivelyinvestigated (Buhmeida et al., 2006, Diagn Pathol. 1:4; Blute et al.,2001, J Urol. 165(1):119-25; Stamey et al., 1999, JAMA.281(15):1395-400; Quinn et al., 2005, Eur J Cancer 41(6):858-87.Review). Recurrence of prostate cancer has been associated with multiplefactors including preoperative PSA level and velocity, clinical stage,Gleason score, level of extracapsular extension, seminal vesicleinvasion, pelvic lymph node status and surgical margin status (Buhmeidaet al., 2006, Diagn Pathol. 1:4; Blute et al., 2001, J. Urol.165(1):119-25; Stamey et al., 1999, JAMA. 281(15):1395-400; Quinn etal., 2005, Eur J Cancer 41(6):858-87. Review). However, due to thesignificant clinical heterogeneity of the disease, these factors are farfrom accurate and the identification of novel biomarkers that are ableto identify patients that are at high risk for cancer progression isthus clearly needed (Harding et al., 1998-1999, Cancer Metastasis Rev.17(4):429-37. Review; Chin et al., 2004, Clin Prostate Cancer.3(3):157-64. Review; Zimmerman et al., 2003, Clin Prostate Cancer.2(3):160-6. Review).

Radiation therapy and surgical resection can be curative in localizeddisease (Roehl et al., 2004, J. Urol. 172(3):910-4; Hull et al., 2002, JUrol. 167(2 Pt 1):528-34; Stephenson et al., 2004, JAMA.291(11):1325-32). However, at present there is no cure for metastaticprostate cancer and the development of molecularly target therapies forprostate cancer is of considerable interest. Tumor vaccines representone type of molecularly target therapy being investigated for prostatecancer (Harada et al., 2003, Int J Clin Oncol. 8(4):193-9. Review;Webster et al., 2005, J Clin Oncol. 23(32):8262-9. Review; Fong et al.,2006, Curr Urol Rep. 7(3):239-46. Review; Madan et al., 2006, Expert RevVaccines. 5(2):199-209. Review). Preclinical and early clinical datasuggest that tumor vaccine therapies are feasible and safe and provideclinical benefits to patients with prostate cancer.

Cancer/Testis (CT) antigens are proteins expressed in normal gametogenictissues and in different types of tumors (Scanlan et al., 2002, ImmunolRev. 188:22-32. Review; Scanlan et al., 2004, Cancer Immun. 4:1. Review;Simpson et al., 2005, Nat Rev Cancer. 5(8):615-25. Review; Zendman etal., 2003, J Cell Physiol. 194(3):272-88. Review). Most CT antigens arehighly immunogenic, eliciting both humoral and cellular immune responsein cancer patients. Due to their restricted expression pattern andimmunogenicity, CT antigens are considered to be ideal targets forcancer immunotherapy (Scanlan et al., 2002, Immunol Rev. 188:22-32.Review; Bodey, 2002, Expert Opin Biol Ther. 2(6):577-84. Review).

As described above, the novel CT antigen, CTSP-1, is exclusivelyexpressed in normal testis and is aberrantly expressed in 47% of tumorcell lines and in 44% of tumors from different histological types(Parmigiani et al., 2006, Proc. Natl. Acad. Sci. U.S.A.103(48):18066-71). CTSP-1 is part of a highly conserved gene family, andantibodies against members of this gene family were detected in 10% ofplasma samples from patients with a wide spectrum of tumors. CTSP-1 wasfound to be aberrantly expressed in 58% of prostate tumors and capableof eliciting a humoral immune response in approximately 20% of prostatecancer patients (Parmigiani et al., 2006, Proc. Natl. Acad. Sci. U.S.A.103(48):18066-71).

Here we confirm that antibodies against CTSP-1 are frequently found inprostate cancer patients, suggesting that this antigen might be apromising candidate for prostate cancer immunotherapy. Moreover,patients presenting antibodies against CTSP-1 had a longer biochemicalrecurrence-free survival, indicating that the presence of a spontaneoushumoral immune response against CTSP-1 could be used as a novelbiomarker for biochemical recurrence in prostate cancer patients withclinically localized disease.

Patients and Methods

Patients and surgical specimens The study included 147 consecutivelocalized prostate cancer patients who underwent radical retropubicprostatectomy with simultaneous bilateral pelvic lymphadenectomy at theUrology Division of the Pelvic Surgery Department of the Hospital doCancer, São Paulo, Brazil, between November 1998 and July 2005. Allcases had clinically localized disease suspected by elevated serum PSAlevels or palpable nodules on a digital rectal examination, and werediagnosed by transrectal ultrasound-guided needle biopsy of theprostate. Serum PSA levels were measured with Elecsys chemiluminescentassay on a Elecsys analyser 1010, Roche®. Patients with PSA levelshigher than 15 ng/mL or Gleason score higher than 7 were submitted tobone scan and patients with positive preoperative imaging were excludedfrom this analysis. Patients with a second non-prostate primary tumour,or that were submitted to radiotherapy before the surgery, were alsoexcluded from this study. Twenty-seven patients received neoadjuvantandrogen suppression.

After RRP, patients were followed up with serum PSA tests every 3 monthsduring the first year, twice a year between the second and the fifthyear and then annually thereafter. Patients with an increase in PSAlevels after RRP were submitted to rectal digital examination, pelvicand abdominal computerized tomography and transrectal ultrasound-guidedneedle biopsy to confirm local recurrence and bone scan to detectdistant metastasis.

Surgical specimens were staged according to the 1997 American JointCommittee on Cancer System and graded using the Gleason system.Specimens were also evaluated for surgical margin status, perineural,angiolymphatic, extracapsular and seminal vesicle invasion as well asfor the presence of metastasis in all removed lymph nodes.Clinicopathological data and follow-up information were obtainedretrospectively from patient's medical records. Patients that did notreturn to the institution for their regular follow-up were contacted bytelephone to update their health status and most recent PSA test. Themean follow up time of this cohort was 52.1 months (range 2.9 to96_(—)2). Biochemical recurrence was defined as a serum PSA level>0.2ng/ml on two consecutive PSA tests after RRP.

Immunoblotting analysis Plasma samples were collected after explicitinformed consent and the study was reviewed and approved by theinstitution's ethical committee. Patient identifiers were removed andsamples were coded to protect confidentiality. Serum antibody responsesagainst the recombinant CTSP-1 protein were tested by standard Westernblot analysis as previously described (Parmigiani et al., 2006, Proc.Natl. Acad. Sci. U.S.A. 103(48):18066-71). Briefly, purified CTSP-1recombinant protein was fractioned on SDS/PAGE gel electrophoresis andtransferred to Hybond-P PVDF membranes. After blocking with PBS solutioncontaining 5% low-fat milk, the membranes were incubated for 1 h at roomtemperature with plasma from prostate cancer patients. Plasma antibodiesbinding to CTSP-1 protein were detected by incubation with goatanti-human IgG HRP-conjugate (Amersham Biosciences) and visualized withECL™ Western Blotting Detection Reagents (Amersham Biosciences).

Statistical Analysis Fisher exact test or Chi-square tests were used toevaluate the association between serum antibody response against CTSP-1and clinicopathological parameters, for continuous variablesMann-Whitney U was used to compare the values between the two groupsconsidering CTSP-1. The preoperative PSA level was considered as acontinuous variable, the pathological Gleason score was divided in twocategorical variables (4 to 6 and 7 to 8) and tumor volume was dividedin 4 categorical variables (1 to 10, 11-30, 31-50 and 51-100).Biochemical recurrence free survival (from the date of the RRP until thedate of the first PSA measurement>0.2 ng/ml or to the date of the lastfollow up) and clinical recurrence free survival (from the date of theRRP until the date of detection of local or distant disease or until thedate of the last follow up) curves were calculated with Kaplan-Meiermethod. Log-rank test was used to assess statistical differences betweenthe curves. Multivariate analysis was carried out using Cox proportionalhazard regression model. All variables presenting p-value<0.20 on theunivariate analysis were selected for building a multiple model(stepwise forward method), since this level of significance eliminatesmany insignificant variables and ensures that all potential explanatoryvariables are included. For all tests, type 1 error (alpha) wasestablished as 0.05 and results were considered statisticallysignificant when p<0.05. All of the statistical analyses were performedusing SPSS Software 10.0 (SPSS Inc., Chicago, Ill.).

Humoral response against CTSP-1 and pathological parameters. Thepathological parameters of the 147 patients analyzed in this study aresummarized in Table 4 below. The mean age of the patients at diagnosiswas 61.6 years (range 41-75) and the mean preoperative PSA level was10.8 ng/ml (range 1.2 to 44). Humoral response against CTSP-1 wasdetected in 37 (25.2%) patients by Western blot.

TABLE 4 Pathological characteristics of the prostate cancer patients.Characteristic Category No. (%) Pathologic T staging T2a 16 (10.9) T2b82 (55.8) T3a 27 (18.4) T3b 14 (9.5) T4 8 (5.4) Pathologic N staging N0145 (98.6) N1 2 (1.4) Gleason score  4-6 98 (66.7)  7-8 49 (33.4) Tumourvolume (%)  1-10 54 (38.8) 11-30 59 (42.4) 31-50 17 (12.2) 51-100 9(6.5) Perineural invasion No 40 (27.4) Yes 106 (72.6) Angiolymphaticinvasion No 22 (15.1) Yes 124 (84.9) Extracapsular invasion No 70 (47.6)Focal 65 (44.2) Diffuse 12 (8.2) Margins Negative 115 (78.2) Positive 32(21.8) Siminal vesical invasion No 134 (91.2) Yes 13 (8.8)

The distribution of pathological parameters according to the presence ofhumoral response against CTSP-1 is listed in Table 5 below. Nostatistically significant association was observed between the presenceof antibodies against CTSP-1 and the following parameters: preoperativePSA level (p=0.173), tumor stage (p=0.126), lymph node status (p=0.409),Gleason score (p=0.139), tumor volume (p=0.478), perineural invasion(p=0.427), angiolymphatic invasion (p=0.427), extracapsular invasion(p=0.319), seminal vesicle invasion (p=0.128) and surgical margins(p=0.344).

TABLE 5 Patient's distribution according to the existence of humoralresponse against CTSP-1. Humoral Response Characteristic No Yes p-valuePathologic T staging T2a 13 (81.3%) 3 (18.7%) T2b 60 (73.2%) 22 (26.8%)T3a 17 (63.0%) 10 (37.0%) 0.126 T3b 14 (100.0%) 0 (0.0%) T4 6 (75.0%) 2(25.0%) Pathologic N staging N0 108 (74.5%) 37 (25.5%) 0.409 N1 2(100.0%) 0 (0.0%) Gleason score  4-6 77 (78.6%) 21 (21.4%) 0.139  7-8 33(67.3%) 16 32.7%) Tumour volume (%)  1-10 40 (74.1%) 14 (25.9%) 11-30 41(69.5%) 18 (30.5%) 0.478 31-50 15 (88.2%) 2 (11.8%) 51-100 7 (77.8%) 2(22.2%) Perineural invation No 28 (70.0%) 12 (30.0%) 0.427 Yes 81(76.4%) 25 (23.6%) Angiolymphatic invasion No 28 (70.0%) 12 (30.0%)0.427 Yes 81 (76.4%) 25 (23.6%) Extracapsular invasion No 55 (78.6%) 15(12.4%) 0.319 Focal 48 (73.8%) 17 (26.2%) Diffuse 7 (58.3%) 5 (41.7%)Margins Negative 84 (73.0%) 31 (27.0%) 0.344 Positive 26 (81.3%) 6(18.7%) Seminal vesicle invasion No 98 (73.1%) 36 (26.9%) 0.128 Yes 12(92.3%) 1 (7.7%)

Humoral response against CTSP-1 and biochemical recurrence. Kaplan-Meieranalysis was then used to estimate the relationship between the presenceof humoral response against CTSP-1 and the risk of biochemicalrecurrence of the disease. The probability of 5-year biochemicalrecurrence-free survival among all of the 147 patients evaluated was61.8%. Patients that did not present antibodies against CTSP-1 had ashorter biochemical recurrence-free interval than did those presentinganti-CTSP-1 antibodies, although the difference between the groups wasnot statistically significant (57.2 versus 75.6%, p=0.075, FIG. 8A).Interestingly, none of the patients presenting antibodies against CTSP-1developed clinical symptoms of the disease (FIG. 8B). Five-yearbiochemical recurrence-free survival in our patient population was alsoinfluenced by the following parameters (Table 6): preoperative PSA level(p<0.001), tumor stage (p<0.001), Gleason score (p=0.016), tumor volume(p<0.001), seminal vesicle invasion (p<0.001) and surgical margins(p<0.001).

TABLE 6 Univariate analysis of biochemical-recurrence-free survival.Characteristic Category 5-year BRFS (%) p-value Pathologic T staging T2a0 T2b 66.6 <0.001 T3a 80.3 T3b 23.6 T4 18.8 Pathologic N staging N0 62.00.341 N1 50.0 Gleason score  4-6 69.1 0.016  7-8 49.9 Tumour volume (%) 1-10 73.0 <0.001 11-30 67.7 31-50 61.5 51-100 22.2 Perineural invasionNo 67.3 0.134 Yes 58.9 Angiolymphatic invasion No 64.0 0.093 Yes 48.5Extracapsular invasion No 73.1 0.161 Focal 54.1 Diffuse 41.6 MarginsNegative 68.4 <0.001 Positive 40.5 Seminal vesicle invasion No 66.9<0.001 Yes 40.5 Preoperative PSA Continuous <0.001 Antibody Positive75.6 0.075 Negative 57.2

Humoral response against CTSP-1 as an independent prognostic marker forbiochemical recurrence. A multivariate analysis was then performed inorder to determine whether the presence of a humoral immune responseagainst CTSP-1 is an independent factor in predicting biochemicalrecurrence of the disease. The preoperative PSA level, the Gleason scoreand the presence of antibodies against CTSP-1 were considered asindependent prognostic factors for biochemical recurrence free survival(Table 7). Patients presenting antibodies against CTSP-1 had a lowerrisk for biochemical recurrence than patients without anti-CTSP-1 (HR0.36, ₉₅%CI 0.2-0.8, p=0.013).

TABLE 7 Multivariate analysis of biochemical-recurrence-free survival.Variables Category HR 95% CI p-value Preoperative PSA Continuous 1.091.06-1.13 <0.001 Antibody Negative 1.0 Ref. 0.013 Positive 0.36 0.2-0.8Gleason score 4-6 1.0 Ref. 0.018 7-8 2.0 1.3-3.7

Discussion

It is widely held that the discovery of novel biomarkers that arecapable of predicting the risk of biochemical recurrence and/or thatcould be used as targets for alternative anti-cancer therapy, such asimmunotherapy, are clearly needed for prostate cancer.

Among various types of epithelial cancers, prostate cancer appears to beone of the best targets for specific immunotherapy. It is generallyrecognized that the human immune system is capable of mounting asignificant immune response against the prostate tissue that can be bothunremitting and destructive in nature (Nelson et al., 2004, J Urol.172(5 Pt 2):S6-11; discussion S11-2. Review). Relevant to this, inverserelationships between the rates of disease recurrence and the number ofinfiltrating T lymphocytes within prostate tumors, as well as, advancedtumor grades and the number of macrophages within prostate tissue, implythat patient cell-mediated immunity may actively suppress prostate tumorprogression (Vesalainen et al., 1994, Eur J Cancer. 30A(12):1797-803;Shimura et al., 2000, Cancer Res. 60(20):5857-61). Moreover, it has beendemonstrated that immunity against normal and cancerous prostate tissuecan be induced by a variety of manipulations of the immune system,including peptide, DNA, virus-based, dendritic cell andgenetically-modified tumor vaccines (Harada et al., 2003, Int J ClinOncol. 8(4):193-9. Review; Webster et al., 2005, J Clin Oncol.23(32):8262-9. Review; Fong et al., 2006, Curr Urol Rep. 7(3):239-46.Review; Madan et al., 2006, Expert Rev Vaccines. 5(2):199-209. Review).In addition, the generally slow progression of prostate cancer providesrelatively long interval of time for the induction and potentiation ofT-cell mediated anti-tumoral immunity during immunotherapeutictreatment. Finally, given that prostate is a relatively dispensableorgan, often removed by surgery as part of prostate cancer treatment,immune response to prostate-related antigens can be considered as tumorspecific in patients with recurrent metastases.

The results of several clinical vaccine trials suggest a clinicalbenefit to patients with prostate cancer and phase III trials arecurrent underway in patients with androgen-independent prostate cancer.The antigens included in these studies are mainly proteins with knownprostate-restricted expression (e.g., PSA, PSMA, PAP) and most of themare not natural targets of an immune response in patients with cancer(Harada et al., 2003, Int J Clin Oncol. 8(4):193-9. Review; Webster etal., 2005, J Clin Oncol. 23(32):8262-9. Review; Fong et al., 2006, CurrUrol Rep. 7(3):239-46. Review; Madan et al., 2006, Expert Rev Vaccines.5(2):199-209. Review). Identifying immunologically recognized proteinsin patients with prostate cancer could thus lead to the development ofother potential vaccine target antigens.

CT antigens represent a novel and expanding family of proteins which areexpressed in normal gametogenic tissues and in different types of tumorsand are also immunogenic in cancer patients (Scanlan et al., 2002,Immunol Rev. 188:22-32. Review; Scanlan et al., 2004, Cancer Immun. 4:1.Review; Simpson et al., 2005, Nat Rev Cancer. 5(8):615-25. Review;Zendman et al., 2003, J Cell Physiol. 194(3):272-88. Review). To date,89 individual CT antigen genes have been described, which are organizedinto 44 families (Simpson et al., 2005, Nat Rev Cancer. 5(8):615-25.Review). It is believed that these antigens escape normal immuneresponse, due to their restricted expression in MHC class I-negativegerm cells (Scanlan et al., 2002, Immunol Rev. 188:22-32. Review; Bodey,2002, Expert Opin Biol Ther. 2(6):577-84. Review). Thus, CT antigenshave been considered to be ideal targets for cancer immunotherapy asthey may serve essentially as tumor-restricted antigens that are at alower risk for preexisting immune tolerance and for creating potentialautoimmune reactions in normal tissues (Scanlan et al., 2002, ImmunolRev. 188:22-32. Review; Bodey, 2002, Expert Opin Biol Ther. 2(6):577-84.Review).

Several members of the CT antigen family are expressed in prostatetumors including PAGE-4, NY-ESO-1, LAGE-1, XAGE-1 and members of theMAGE-A family. However, little is known about their ability to inducehumoral and cellular immune responses in prostate cancer patients andtheir utility as target for prostate cancer immunotherapy remains poorlyunexplored (Prikler et al., 2004, Aktuelle Urol. 35(4):326-30; Nakada etal., 2003, Cancer Immun. 3:10; Lethe et al. 1998, Int J Cancer.76(6):903-8; Egland et al., 2002, Mol Cancer Ther. 1(7):441-50; Kufer etal., 2002, Cancer Res. 62(l):251-61).

As shown above, a novel CT antigen named CTSP-1, is exclusivelyexpressed in normal testis among normal tissues and is aberrantlyexpressed in 58% of prostate tumors. CTSP-1 is part of a highlyconserved gene family, and antibodies against members of this genefamily were detected in approximately 20% of prostate cancer patients(Parmigiani et al., 2006, Proc. Natl. Acad. Sci. U.S.A.103(48):18066-71). In this study, a natural humoral immune responseagainst CTSP-1 was detected in 25.2% of the patients with clinicallylocalized prostate cancer. Since the antibodies detected against CTSP-1are of the IgG class, the activation of CD4+ T-cell response to CTSP-1could also be present in these patients. Further experiments are ongoingto test if the CTSP-1 protein is able to induce CD4+ and CD8+ T-cellresponses in cancer patients. These results confirm our initialobservations and suggest that CTSP-1 might be a promising candidate forprostate cancer immunotherapy.

Here, we demonstrated that the presence of antibodies against CTSP-1 isan independent prognostic predictor of biochemical recurrence after RRP.Reports on the association of expression and the presence of immuneresponse against CT antigens with clinical events and prognosis arelimited to a few studies involving a limited number of patients. Most ofthese studies are on the highly immunogenic CT antigen NY-ESO-1.NY-ESO-1 mRNA and/or protein expression was predominantly detected inhigher grade and advanced breast and esophageal tumors, transitionalcell bladder carcinomas and medullary thyroid carcinomas, however noassociation with disease outcome was established. Similarly,immunological assessment of patients with NY-ESO-1 positive tumorsrevealed no association with clinicopathological parameters and diseaseoutcome (Sharma et al., 2003, Cancer Immun. 3:19; Sugita et al., 2004,Cancer Res. 64(6):2199-204; Maio et al., 2003, J Clin Endocrinol Metab.88(2):748-54; Akcakanat et al., 2004, Cancer Chemother Pharmacol.54(1):95-100). In prostate cancer, NY-ESO-1 protein was predominantlyexpressed in hormone-refractory prostate tumors when compared tolocalized tumors and anti-NY-ESO-1 antibodies were also more frequentlyfound in the serum of patients with advanced disease. Spontaneousserological response against NY-ESO-1 was associated with poor survival(Fossa et al., 2004, Prostate. 59(4):440-7). Together with our own data,these results suggests that the presence of a natural immune responseagainst CT antigens could be used as prognostic factors, specially inprostate cancer, and deserves further evaluation.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: (a) a nucleic acid molecule that comprises anucleotide sequence encoding the amino acid sequence of SEQ ID NO:20,and (b) a full-length complement of (a).
 2. The isolated nucleic acidmolecule of claim 1, wherein the isolated nucleic acid moleculecomprises the nucleotide sequence set forth as SEQ ID NO:8.
 3. Theisolated nucleic acid molecule of claim 2, wherein the isolated nucleicacid molecule consists of the nucleotide sequence set forth as SEQ IDNO:8.
 4. A composition comprising the isolated nucleic acid molecule ofclaim 1 and a carrier.
 5. A composition comprising the isolated nucleicacid molecule of claim 1 attached to a solid substrate.
 6. A kitcomprising: one or more nucleic acid molecules as set forth in claim 1,wherein the one or more nucleic acid molecules are detectably labeled orare bound to a solid substrate.
 7. The kit of claim 6, wherein the oneor more nucleic acid molecule are detectably labeled.
 8. The kit ofclaim 6, wherein the one or more nucleic acids are bound to a solidsubstrate.
 9. An expression vector comprising the isolated nucleic acidmolecule of claim 1 operably linked to a promoter.
 10. An isolated hostcell transformed or transfected with the expression vector of claim 9.